Studies on the Fc gamma receptor of the murine macrophage-like cell line P388D1. II. Binding of human IgG subclass proteins and their proteolytic fragments.

Binding studies of human IgG proteins to murine P388D1 cells indicated that they bind to an apparently homogeneous Fc receptor population. The association constant was 0.89 x 10(6)M-1 at 22 degrees C and was comparable to the binding affinities of homologous murine IgG2a and IgG2b. The number of receptor sites was found to be approximately 6 x 10(5)/cell. Fc gamma 1 and Fc gamma 3 fragments bound with an affinity comparable to that of the parent proteins. The P388D1 receptors could discriminate between the human IgG subclasses; the relative cytophilic activity was IgG3 greater than IgG1 greater than IgG4 and IgG2 was devoid of binding activity. Fragments corresponding to the C gamma 2 and C gamma 3 domains of human IgG1 were both unable to bind to the P388D1 receptors either alone or in equimolar combination. This suggests that the cytophilic site may be formed cooperatively by interaction between the two domains. The integrity of the hinge region appeared to be essential for full expression of cytophilic activity since reduction of the hinge-region disulfides in both human IgG1 and its Fc fragment markedly decreased their binding affinity. In addition, a mutant IgG1 molecule lacking the hinge region was significantly less cytophilic than its normal counterpart.     

Enhancement of macrophage immune and nonimmune receptor-mediated phagocytosis by a low molecular weight soluble factor from resident thymocytes

Macrophage phagocytic activity is regulated in part by products of activated T lymphocytes. We previously reported that a heat-stable soluble factor derived from resident (nonactivated) thymocytes increases murine peritoneal macrophage Fc-dependent phagocytosis. In the present study, we further investigate the effect of the thymocyte factor on immune and nonimmune receptor-mediated phagocytosis, Fc receptor expression, and its approximate m.w. After 4 days of incubation, cellfree thymocyte supernatant produced a mean (three experiments) 2.10-, 2.08-, and 1.97-fold increase in macrophage phagocytosis of C3-, IgG-, and tannic acid-treated erythrocytes, respectively. Macrophage IL 1 production was not enhanced by a similar concentration of thymocyte supernatant. The thymocyte factor(s) increased the number of IgG2a Fc receptors (FcRI) from 2.4 x 10(5) to 3.8 x 10(5) receptor sites per macrophage. The number of Fc receptors that bind IgG1 and IgG2b (FcRII) was not altered. The soluble factor(s) that increased Fc-mediated phagocytosis passed through both 6000- to 8000-dalton and 2000-dalton cutoff dialysis membranes and eluted from a Sephadex G-25 Fine column over a m.w. range of 200 to 1000 daltons, with a peak activity at 450 daltons. These data suggest that resident thymocytes enhance macrophage phagocytosis of opsonized and nonopsonized particles through the elaboration of a low m.w. substance(s).     

Protein kinase activity associated with Fc gamma 2a receptor of a murine macrophage like cell line, P388D1

The properties of protein kinase activity associated with Fc receptor specific for IgG2a (Fc gamma 2aR) of a murine macrophage like cell line, P388D1, were investigated. IgG2a-binding protein isolated from the detergent lysate of P388D1 cells by affinity chromatography on IgG-Sepharose was found to contain four distinct proteins of Mr 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with [gamma-32P]ATP. The autophosphorylation of Fc gamma 2a receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Casein was found to be a much better phosphate acceptor than histone in this system, as casein incorporated about 32-fold more 32P than histone did. Phosphorylation of casein catalyzed by Fc gamma 2a receptor complex was dependent on casein concentration (maximum phosphate incorporation being at 0.5 mg/mL), increased with time or temperature, was dependent on the concentration of ATP and Mg2+, and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn2+ (greater than 25 mM) or KCl (greater than 100 mM) or by a small amount of heparin (greater than 10 units/mL) and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fc gamma 2a receptor used ATP as substrate with an apparent Km of 2 microM as well as GTP with an apparent Km of 10 microM. Prior heating (60 degrees C for 15 min) or treatment with protease (trypsin or Pronase) of Fc gamma 2a receptor complex almost totally abolished casein kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Modulation of human polymorphonuclear leukocyte IgG Fc receptors and Fc receptor-mediated functions by IFN-gamma and glucocorticoids

Human polymorphonuclear neutrophils (PMN) normally express two distinct types of IgG Fc gamma R, the 40-kDa Fc gamma R referred to as Fc gamma RII and the low affinity 50- to 70-kDa Fc gamma R designated Fc gamma RIII. A third type of Fc gamma R, the 72-kDa high affinity receptor known as Fc gamma RI, is also detectable on PMN that have been activated by IFN-gamma. Using mAb that discriminate among the three known types of Fc gamma R, we examined the effects of IFN-gamma and glucocorticoids on human PMN Fc gamma R expression. We also studied effects of IFN-gamma and the synthetic glucocorticoid dexamethasone (DEX) on antibody-dependent cytotoxicity (ADCC) of chicken erythrocytes and phagocytosis of IgG-coated ox RBC by human PMN. In 20 donors studied, we found that treatment of PMN with 400 U/ml IFN-gamma induced a 9- to 20-fold increase in the number of Fc gamma RI sites per cell, and DEX inhibited this induction of Fc gamma RI by 39 to 73%. Similarly, DEX significantly reduced the IFN-gamma stimulation of ADCC and phagocytosis. IFN-gamma had no effect on expression of Fc gamma RII or Fc gamma RIII. Fc gamma RI and Fc gamma RII expression was unaltered by 24 h of treatment with DEX alone, but Fc gamma RIII expression was sometimes increased by about 20% on PMN cultured with DEX. Nevertheless, we found a small but significant inhibition of ADCC and phagocytosis by 200 nM DEX. Our results indicate that Fc gamma RI plays a major but not exclusive role in the regulation of ADCC and phagocytosis by IFN-gamma and DEX.     

Requirements for the opsonic activity of human IgG directed to type 6 group A streptococci: net basic charge and intact Fc region

By two independent techniques for separating human opsonic IgG for group A type 6 streptococci into fast- and slow-migrating fractions, it was found that the opsonic activity was localized within the basic charge population. This charge dependence was found to be a characteristic of the IgG isolated from three individuals. When the fast- and slow-migrating IgG fractions were tested for their ability to bind to purified M6 protein, antibodies in both opsonic and nonopsonic populations exhibited binding activity, with the majority being located within the opsonic IgG in two of the three individuals; the third displayed greater binding in the nonopsonic population. The functional difference observed in the antibody populations to this M antigen may be a reflection of the net charge within the area of the antibody binding site, which suggests that the opsonic antibodies need to bind to acidic residues along the outer surface of the fibrillar M protein molecule. F(ab')2 fragments prepared from both human and rabbit type 6 opsonic IgG were still able to bind to the M6 molecule but were unable to mediate opsonization of type 6 streptococci. However, the F(ab')2 fragments had the capacity to enhance or amplify the opsonic activity of low concentrations of opsonic IgG molecules. The results suggest that the M protein molecule may function as an active inhibitor of phagocytosis and that F(ab')2 fragments from opsonic IgG have the capacity to neutralize the "active" determinants on the molecule, thus allowing lower concentrations of IgG with functional Fc receptors to mediate phagocytosis.     

Binding of IgG-opsonized particles to Fc gamma R is an active stage of phagocytosis that involves receptor clustering and phosphorylation

Fc gammaR mediate the phagocytosis of IgG-coated particles and the clearance of IgG immune complexes. By dissecting binding from internalization of the particles, we found that the binding stage, rather than particle internalization, triggered tyrosine phosphorylation of Fc gammaR and accompanying proteins. High amounts of Lyn kinase were found to associate with particles isolated at the binding stage from J774 cells. PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), an Src kinase inhibitor, but not piceatannol, an inhibitor of Syk kinase, reduced the amount of Lyn associated with the bound particles and simultaneously diminished the binding of IgG-coated particles. Studies of baby hamster kidney cells transfected with wild-type and mutant Fc gammaRIIA revealed that the ability of the receptor to bind particles was significantly reduced when phosphorylation of the receptor was abrogated by Y298F substitution in the receptor signaling motif. Under these conditions, binding of immune complexes of aggregated IgG was depressed to a lesser extent. A similar effect was exerted on the binding ability of wild-type Fc gammaRIIA by PP2. Moreover, expression of mutant kinase-inactive Lyn K275R inhibited both Fc gammaRIIA phosphorylation and IgG-opsonized particle binding. To gain insight into the mechanism by which protein tyrosine phosphorylation can control Fc gammaR-mediated binding, we investigated the efficiency of clustering of wild-type and Y298F-substituted Fc gammaRIIA upon binding of immune complexes. We found that a lack of Fc gammaRIIA phosphorylation led to an impairment of receptor clustering. The results indicate that phosphorylation of Fc gammaR and accompanying proteins, dependent on Src kinase activity, facilitates the clustering of activated receptors that is required for efficient particle binding.     

Macrophage cell cycling: influence on Fc receptors and antibody-dependent phagocytosis

The influence of cell cycling on the density and binding properties of IgG2a Fc receptors and their associated antibody-dependent phagocytic activity was investigated with the P388D1 murine macrophage cell line. Unseparated macrophages and subpopulations of elutriated macrophages, enriched for cells in G1, S, and G2 + M phases were compared to detect possible differences in IgG2a-dependent phagocytosis. Suspensions of G2 + M phase cells were appreciably enhanced in phagocytic activity over G1-phase cells, which were less phagocytic than unseparated macrophage populations. An analysis of the binding of 125I-IgG2a myeloma protein disclosed that the IgG2a Fc receptor avidity remained essentially unchanged during cell cycle traverse, whereas the number of IgG2a Fc receptors more than doubled as cells cycled from G1 to G2 + M (1.5 X 10(5) vs 3.4 X 10(5) receptors per cell). With their increased size relative to G1 cells, and the resultant increase in receptor number, G2 phase cells should have more productive collisions with the antibody-coated target cells and greater phagocytic capacity.     

Internalization and degradation of macrophage Fc receptors during receptor-mediated phagocytosis

Macrophage receptors for the Fc domain of immunoglobulin G (IgG) can mediate the efficient binding and phagocytosis of IgG-coated particles. After internalization, phagocytic vacuoles fuse with lysosomes, initiating the degradation of their contents. Using specific monoclonal and polyclonal antireceptor antibodies, we have now analyzed the internalization and fate of Fc receptors during the uptake of IgG- coated erythrocytes and erythrocyte ghosts by mouse peritoneal macrophages. Receptor-mediated phagocytosis led to the selective and largely irreversible removal of Fc receptors (greater than 50%) from the macrophage plasma membrane. The expression of several other plasma membrane proteins (including a receptor for complement), recognized by a series of antimacrophage monoclonal antibodies, was affected only slightly. Interiorized Fc receptors were rapidly and selectively degraded. This was demonstrated by a series of turnover studies in which Fc receptor was immunoprecipitated from lysates of 125I-labeled macrophages. These experiments were made possible by the development of a polyclonal rabbit antiserum, raised against isolated Fc receptor, which recognized the receptor even in the presence of bound ligand. In control cells, the receptor turned over with a t1/2 of approximately 10 h; after phagocytosis, greater than 50% of the receptors were degraded with a t1/2 of less than 2 h. The turnover of other unrelated plasma membrane proteins was unaffected (t1/2 of 18-23 h) under these conditions.     

Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions

Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complementenhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens.Key words: antibody, Fc, complement, CDC, C1q, ADCC, phagocytosis, CD20, CD19, CD40     

The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. I. Identification of the population of effector cells.

Mononuclear cells (MC) from human blood were fractionated by a variety of physical and immunologic techniques, and the cellular subpopulations generated were assessed for their capacity to lyse herpes simplex virus (HSV)-infected target cells in the presence of IgG antibody to HSV. Latex phagocytosis and surface marker studies were performed in parallel in order to identify the major effector cells by their phagocytic properties and their possession of surface immunoglobulin and receptors for either sheep erythrocytes, C3, or the Fc fragment of IgG. Cytotoxic effector cell activity was unaffected or slightly enhanced after the removal of plastic-adherent or carbonyl iron-adherent MC, indicating that the major effector cell is not a classical monocyte. Similar results were obtained after removal of more than 90% of the T cells by depletion of rosette-forming cells. Likewise, effector cell activity was generally unchanged when more than 95% of the B cells were removed by filtering MC on nylon wool columns. Effector cell function was also found to be normal in three patients with B cell-deficient X-linked agammaglobulinemia. These observations strongly suggest that the effector cells are not T cells or B cells. A 4- to 5-fold enrichment in effector cells, however, was consistently found in a subpopulation, consisting of 5% of the unfractionated MC, that was dramatically enriched both for nonphagocytic cells with only Fc receptor (K cells) and for nonphagocytic cells with no detectable surface markers (null cells). Since, as is demonstrated in the accompanying report, effector surface Fc receptors play a critical role in the mediation of antibody-dependent cellular cytotoxicity directed at HSV-infected target cells, the major mononuclear effector cell in human blood is a K cell.     

Concentration-dependent effect of fibrinogen on IgG-specific antigen binding and phagocytosis

In this paper, we aim to characterize fibrinogen-IgG interactions, and explore how fibrinogen alters IgG-mediated phagocytosis.Using enzyme-linked binding assays, we found that fibrinogen binding to IgG is optimized for surfaces coated with high levels of IgG. Using a similar method, we have shown that for an antigen unable to specifically bind fibrinogen, fibrinogen enhances binding of antibodies towards that antigen. For binding of IgG antibodies to cells expressing Fc receptors, we found a bimodal binding response, where low levels of fibrinogen enhance binding of antibody to Fc receptors and high levels reduce it. This corresponds to a bimodal effect on phagocytosis of IgG-coated particles, which is inhibited in the presence of excess IgG during coating of the particles with antibodies and fibrinogen.We conclude that fibrinogen can modulate phagocytosis of IgG-coated particles in vitro by changing IgG binding behavior, and that high fibrinogen levels could negatively affect phagocytosis.     

Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions

Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complement-enhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential, and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens.     

Introduction of purified hexosaminidase A into Tay-Sachs leukocytes by means of immunoglobulin-coated liposomes.

To determine whether ligand-receptor interactions could engender the selective uptake by deficient cells of enzyme-laden liposomes, aggregated human IgG was used to coat liposomes which had previously trapped purified hexosaminidase A (Hex A). By a new, high-yield procedure, Hex A was purified 7000-fold from human placenta: the homogeneous protein had a pI of 5.4, permitting nonelectrostatic trapping in the aqueous interstices of anionic multilamellar liposomes (molar ratios of phosphatidyl-choline-dicetyl phosphate-cholesterol, 7:2:1). Trapped Hex A was separated from free enzyme by means of Sephadex G-200 chromatography: 1.3 +/- 0.3 mUnits of Hex A/mumol of phospholipid became associated with liposomes and trapped glucose, utilized as a marker of the aqueous compartment. Once sequestered, the enzyme remained latent until lamellae were disrupted by Triton X-100. Presence of enzyme in aqueous compartments was proved by the demonstration of increased trapping (0.02-1.33 mUnits/mumol of phospholipid) with increments in like-sign repulsion of the bilayers produced by increasing molar ratios of anionic dicetyl phosphate (5-20%). To provide for ligand-receptor interaction with surface Fc receptors of human polymorphonuclear leukocytes (PMN's), liposomes were coated by heat-aggregated (62 degrees C, 10 min) human IgG. PMN's from Tay-Sachs patients genetically deficient in Hex A activity readily incorporated exogenous Hex A provided in this fashion. PMN's exposed to enzyme-laden liposomes coated with aggregated IgG incorporated significantly more Hex A than when the enzyme was presented in uncoated liposomes or in liposomes coated with native IgG, which engages Fc receptors with less avidity. Free enzyme was not endocytized. Acquisition of specific Hex A isozyme activity by cells (determined by DEAE-cellulose chromatography) was not due to surface adsorption since cytochalasin B, which prevents phagocytosis but not surface adherence; blocked uptake. Incorporation of the isozyme by deficient cells was also demonstrated by starch gel electrophoresis, and ultrastructural studies showed that the immunoglobulin-coated, Hex A-containing liposomes were taken up into PMN lysosomes after membrane fusion. The studies indicate that liposomes coated with surface ligands may be used to introduce enzyme or other materials into deficient cells possessing appropriate surface receptors.     

Augmentation of macrophage complement receptor function in vitro. V. Studies on the mechanisms of ligation of macrophage Fc receptors required to trigger macrophages to signal T lymphocytes to elaborate the lymphokine that activates macrophage C3 receptors for phagocytosis

We previously described a unique lymphokine that activates macrophage C3 receptors for phagocytosis. The lymphokine is generated when T lymphocytes receive a signal from macrophages that have ingested IgG-coated material. In the present work, we examined the mechanisms by which macrophage Fc receptors must be engaged for macrophages to signal lymphocytes to elaborate the lymphokine. We found that ingestion mediated by any of the three classes of murine macrophage Fc receptors was sufficient to trigger macrophages, and that engagement of macrophage Fc receptors by immobilized immune complexes was effective as well. We also found that ligation of Fc receptors by an anti-Fc receptor IgG antibody or by its F(ab')2 or Fab fragments also triggered macrophages. The ability of monovalent ligation of the receptor to elicit biologic activity suggests that this system may be of value in elucidating general mechanisms by which ligand binding of receptors is transduced into biologic effects.     

Defective mononuclear phagocyte function in systemic lupus erythematosus: dissociation of Fc receptor-ligand binding and internalization

Fc receptor-mediated mononuclear phagocyte system (MPS) clearance is impaired in systemic lupus erythematosus (SLE) and may contribute to the pathogenesis of the immune complex disease. To investigate the basis of MPS dysfunction, we have examined concurrent in vivo and in vitro Fc receptor function in 22 patients with SLE and 23 disease-free adults. Blood monocyte Fc receptor binding was increased rather than decreased as predicted by the saturation hypothesis of MPS blockade. Rosette formation of IgG-sensitized bovine erythrocytes (EA) with monocytes demonstrated increased Fc receptor-ligand binding in SLE (percent rosettes: 40 +/- 12 vs 27 +/- 8, p less than 0.001). Scatchard analysis of the binding of radiolabeled IgG oligomers to SLE monocytes indicated a mean receptor number 30% higher than control, although this did not reach statistical significance. Despite enhanced Fc receptor-ligand (EA) binding, Fc-mediated phagocytosis of EA was decreased in SLE (1.7 +/- 0.7 erythrocytes/monocytes/hour vs 2.6 +/- 1.0, p less than 0.004). This decrease in phagocytosis by blood monocytes from SLE patients was significantly greater than that attributable to the predominance in SLE of individuals with certain HLA B cell alloantigens and intrinsically lower phagocytic rates (p less than 0.05 for all groups). This decrease therefore represents a disease-acquired characteristic. Furthermore, the phagocytic rate of the four SLE patients with marked prolongation in MPS clearance was significantly lower than that of the eight patients with near normal clearance values (p less than 0.01). Saturation of Fc receptors by immune complexes does not explain impaired immune clearance in SLE. Our results indicate that despite increased binding of the EA ligand, Fc receptor-mediated phagocytosis is markedly impaired in SLE monocytes. This impairment cannot be explained on the basis of HLA-related differences in phagocytosis among lupus patients. The defect in phagocytosis of EA is most profound in those patients with the most significantly impaired MPS clearance. Thus, the dissociation of receptor-ligand binding and receptor-mediated internalization may contribute significantly to the in vivo clearance defect in SLE.     

Fcγ receptors exhibit different phagocytosis potential in human neutrophils

In neutrophils, two receptors for IgG antibodies, namely FcγRIIA and FcγRIIIB are constitutively expressed, and a third one, FcγRI, can be upregulated by interferon-γ. Whether FcγRIIIB is capable of triggering phagocytosis by itself is still controversial. The main role of FcγRI has not been clearly established in these cells. To address this problem, neutrophils were treated with interferon-γ, and then phagocytosis mediated by each type of Fcγ receptor was evaluated by flow cytometry. FcγRIIA was the most efficient receptor for phagocytosis. FcγRIIIB could mediate phagocytosis but much less efficiently than FcγRIIA. Both FcγRIIA- and FcγRIIIB-mediated phagocytosis were blocked by inhibitors of Src family kinases, Syk, PI 3-K, and ERK. In contrast, interferon-γ-induced FcγRI was not able to mediate phagocytosis. Also, FcγRI did not activate ERK in the nucleus, but was however able to stimulate an efficient calcium rise. These data show that different neutrophil Fcγ receptors possess different phagocytosis capabilities: FcγRIIA and FcγRIIIB, but not FcγRI, promote phagocytosis.     

Biological Activities of Murine Low-Affinity Fc Receptors for IgG

Murine low-affinity Fc receptors for IgG (FcγRIIbl, FcγRIIb2, and FcγRIII) bind the same IgG subclasses and are not distinguished by available anti-FcγRII/III mAbs (2.4G2). They trigger various biological activities, among which are the internalization of soluble and particulate immune complexes, cell activation, and its regulation. To determine the biological properties of the three murine receptors, each was expressed by stable transfection of corresponding cDNAs in two model cells: the murine lymphoma B cell IIA1.6 and the rat basophilic leukemia cell RBL-2H3. Biological activities of recombinant receptors were triggered with soluble immune complexes or 2.4G2 IgG in IIA1.6 cells, which express no FcγR, and with 2.4G2 Fab or F(ab′)₂, cross-linked with mouse anti-rat F(ab′)₂ in RBL, which express rat FcγR. Conditions for studying cell activation and endocytosis in both cell models are described, as are conditions for studying phagocytosis in RBL cells and antigen presentation or regulation of cell activation in IIA1.6 cells. Internalization of immune complexes was triggered by FcγRIIb2 and FcγRIII, but not by FcγRIIb1. Intracytoplasmic sequences required for phagocytosis and endocytosis could be distinguished in FcγRIIb2, but not in FcγRIII. Cell activation was restricted to FcγRIII. FcγRIII-mediated endocytosis, phagocytosis, and cell activation involved the consensus tyrosine-containing activation motif found in the intracytoplasmic domain of the γ subunit. Regulation of cell activation was induced by both FcγRII isoforms and depended on the same sequence as endocytosis. As a consequence, a single motif can determine more than one biological response of the cell, and a given response may be triggered by several motifs, borne by different FcγR.     

Superoxide anion production from guinea pig macrophages stimulated with immune complexes of different IgG subclasses

Guinea pig peritoneal macrophages produced superoxide anions (O2-) when reacted with ovalbumin complexes of homologous IgG1 and IgG2 antibodies. In this reaction, IgG2 complexes were about three times as active as IgG1 complexes. But the susceptibility of IgG1 complexes to phagocytosis by the cells appeared to be indistinguishable from that of IgG2 complexes. The avidity of IgG1 complexes in the antigen excess zone for Fc receptors on the cells was lower than that of the IgG2 counterparts. The amount of IgG1 complex bound to the cells, however, did not significantly differ from that of IgG2 complexes when compared using each complex at the equivalence zone which showed maximal effector functions on the cells. The binding of Clq to IgG2 complexes increased markedly the amounts of complexes bound to the cells, but it reduced O2- generation. These results suggest that the difference in abilities of IgG1 and IgG2 complexes to promote O2- generation may be caused by different structures of the Fc parts or their antigen complexes involved in priming macrophages for O2- generation.     

Plasticity of Human THP–1 Cell Phagocytic Activity during Macrophagic Differentiation

Studies of the role of macrophages in phagocytosis are of great theoretical and practical importance for understanding how these cells are involved in the organism’s defense response and in the development of various pathologies. Here we investigated phagocytic plasticity of THP–1 (acute monocytic human leukemia) cells at different stages (days 1, 3, and 7) of phorbol ester (PMA)–induced macrophage differentiation. Analysis of cytokine profiles showed that PMA at a concentration of 100 nM induced development of the proinflammatory macrophage population. The functional activity of macrophages was assessed on days 3 and 7 of differentiation using unlabeled latex beads and latex beads conjugated with ligands (gelatin, mannan, and IgG Fc fragment) that bind to the corresponding specific receptors. The general phagocytic activity increased significantly (1.5–2.0–fold) in the course of differentiation; phagocytosis occurred mostly through the Fc receptors, as shown previously for M1 macrophages. On day 7, the levels of phagocytosis of gelatin-and Fc–covered beads were high; however, the intensity of ingestion of mannan–conjugated beads via mannose receptors increased 2.5–3.0–fold as well, which indicated formation of cells with an alternative phenotype similar to that of M2 macrophages. Thus, the type and the plasticity of phagocytic activity at certain stages of macrophage differentiation can be associated with the formation of functionally mature morphological phenotype. This allows macrophages to exhibit their phagocytic potential in response to specific ligands. These data are of fundamental importance and can be used to develop therapeutic methods for correcting the M1/M2 macrophage ratio in an organism.