Effect of IgG for intravenous use on Fc receptor-mediated phagocytosis by human monocytes.

Polyspecific IgG given intravenously at high doses (IVIG) is used for immunomodulatory therapy in autoimmune diseases such as idiopathic thrombocytopenic purpura and myasthenia gravis. It is assumed that the clinical effect is brought about in part by a modulation of mononuclear phagocyte function, in particular by an inhibition of Fc receptor (FcR) mediated phagocytosis. In the present study, the effect of IVIG on FcR-mediated phagocytosis by monocytes was analysed in vitro. Since monocytes exposed to minute amounts of surface-bound IgG displayed impaired phagocytosis of IgG-coated erythrocytes (EA), the effect of IVIG was studied with mononuclear cells suspended in teflon bags in medium containing 10% autologous serum and IVIG (2-10 mg/ml). Monocytes pre-exposed to IVIG and then washed, displayed impaired ingestion of EA when compared with control cells cultured in 10% autologous serum only. The decrease in phagocytosis was observed with sheep erythrocytes treated with either rabbit IgG or bovine IgG1 and with anti-D-treated human erythrocytes. This suggests that phagocytosis via both FcR type I (FcRI) and type II (FcRII) was decreased. The impairment of phagocytosis was dependent on the presence of intact IgG and was mediated by IVIG from nulliparous donors and from multigravidae to the same extent, suggesting that alloantibodies contained in IVIG have a minor role in modulating FcR-mediated phagocytosis by monocytes. A flow cytometric analysis using anti-FcRI, FcRII and FcRII monoclonal antibodies showed that IVIG treatment upregulated FcRI expression but did not significantly alter the expression of FcRII and FcRIII.     

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.     

Effect of soluble immune complexes of Fc and C3 receptor-dependent phagocytosis by human monocytes.

The capacity was studied of bovine serum albumin (BSA) rabbit anti-BSA and ovalbumin (OA) rabbit anti-OA immune complexes of different composition to inhibit the Fc receptor-dependent adherence and phagocytosis of sensitized sheep red blood cells by human monocytes. Parallel experiments were performed on the ability of the immune but complement-reacted complexes to inhibit the C3 receptor-dependent phagocytosis of C3-coated yeast particles. The extent of inhibition of both the Fc and C3 receptor-dependent phagocytosis was proportional to the antibody avidity of the complex used. Immune complexes made at equivalence and at moderate antibody excess markedly inhibited both types of phagocytosis, whereas those made at moderate antigen excess had only a weak inhibitory effect. These findings can be explained by the correlation between the Fc receptor-binding and complement-activating capacities of immune complexes of different composition. An alternative explanation, however, is also discussed.     

Bispecific anti-human red blood Rhesus-D antigen x anti Fc gamma RI targeted antibody-dependent cell-mediated cytotoxicity and phagocytosis by mononuclear leucocytes.

The Fc receptor mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis induced by bispecific antibody (BsAb) to the high-affinity Fc receptor for IgG (Fc gamma RI) and to human red blood group antigen RhD were studied in vitro, using human mononuclear leucocytes as effector cells. The results were compared with those obtained by using a human monoclonal IgG1 anti-RhD used alone and a reference human polyclonal anti-RhD antibody. The effect of non-specific human IgG on FcR-mediated functions by mononuclear leucocytes was checked. The results demonstrate that BsAb presents a high resistance of Fc-mediated function to blockade by non-specific human IgG compared with that of both polyclonal and monoclonal anti-RhD antibodies. These results further encourage possible clinical application of bispecific antibody in passive immunotherapy.     

Protein tyrosine kinase activity is essential for Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes.

Our previous study revealed that the intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma receptor I (Fc gamma RI) or Fc gamma RII is a phospholipase C (PLC)-dependent process. The aim of the present study was to investigate whether protein tyrosine kinase (PTK) activity plays a role in the Fc gamma R-mediated intracellular killing of bacteria and activation of PLC in these cells. The results showed that phagocytosis of bacteria by monocytes was not affected by the PTK inhibitors genistein and tyrphostin-47. The intracellular killing of S. aureus by monocytes after cross-linking Fc gamma RII or Fc gamma RII with anti-Fc gamma R monoclonal antibody and a bridging antibody or with human immunoglobulin G (IgG) was inhibited by these compounds in a dose-dependent fashion. The production of O2- by monocytes after stimulation with IgG or IgG-opsonized S. aureus was almost completely blocked by the PTK inhibitor. These results indicate that inhibition of PTK impairs the oxygen-dependent bactericidal mechanisms of monocytes. Genistein and tyrphostin-47, which do not affect the enzymatic activity of purified PLC, prevented activation of PLC after cross-linking Fc gamma RI or Fc gamma RII, measured as an increase in the intracellular inositol 1,4,5-trisphosphate concentration. Cross-linking Fc gamma RI or Fc gamma RII induced rapid tyrosine phosphorylation of several proteins in monocytes, one of which was identified as PLC-gamma 1, and the phosphorylation could be completely blocked by PTK inhibitors, leading to the conclusion that activation of PLC after cross-linking Fc gamma R in monocytes is regulated by PTK activity. Together, these results demonstrate that PTK activity is essential for the activation of PLC which is involved in the Fc gamma R-mediated intracellular killing of S. aureus by human monocytes.     

Neonatal Fc receptor induces intravenous immunoglobulin growth suppression in Langerhans cell histiocytosis

The neonatal Fc receptor (FcRn) plays a role in trafficking IgG and albumin and is thought to mediate intravenous immunoglobulin (IVIG) therapy for certain diseases. IVIG can be used for the treatment of human Langerhans cell histiocytosis (LCH); however, the mechanism remains unclear. The expression and function of FcRn protein have not been studied in LCH, though the expression of FcRn messenger RNA (mRNA) have been reported. In this report, we confirmed the expression of FcRn in 26 of 30 pathological cases (86.7%) diagnosed immunohistochemically as LCH. The expression was independent of age, gender, location, multi- or single-system, and the status of BRAFV600E immunostaining. We also confirmed the expression of FcRn mRNA and protein in the human LCH-like cell line, ELD-1. FcRn suppressed albumin consumption and growth of IVIG preparation-treated ELD-1 cells, but not of IVIG preparation-untreated or FcRn-knockdown ELD-1 cells. In addition, FITC-conjugated albumin was taken into Rab11-positive recycle vesicles in mock ELD-1 cells but not in FcRn-knockdown ELD-1 cells. IVIG preparation prolonged this status in mock ELD-1 cells. Therefore, ELD-1 recycled albumin via FcRn and albumin was not used for metabolism. Our results increase our understanding of the molecular mechanism of IVIG treatment of LCH.     

Fc gamma RII-mediated superoxide production by phagocytes is augmented by GM-CSF without a change in Fc gamma RII expression

Freshly purified neutrophils and monocytes respond to multiple cross-linking of Fc gamma RII with the IgG1 monoclonal antibody, CIKM5, with a rapid rise in Ca(2+)i, but not with a respiratory burst, although superoxide is generated by these cells when stimulated with the chemotactic peptide, FMLP, or phorbol ester (TPA). Incubation in vitro for 30-60 min at 37 degrees C in medium + 0.1% FCS had no effect on the neutrophil superoxide response to CIKM5 but induced a weak monocyte response in 11/13 experiments. However, incubation with rhGM-CSF (10 ng/ml) under similar conditions induced a neutrophil respiratory burst in response to cross-linking Fc gamma RII in 12/14 experiments and enhanced the monocyte response by 181%. GM-CSF also enhanced the response of neutrophils and monocytes to FMLP by 308% and 165%, respectively. The response to TPA was not significantly enhanced by GM-CSF. rhIFN-gamma (100 mu/ml) was ineffective as a priming agent for all agonists tested in short-term incubations but augmented the monocyte response to CIKM5 after 5 d exposure in vitro. Whilst GM-CSF induced neutrophil superoxide production in response to cross-linking Fc gamma RII, there was no concomitant change in Fc gamma RII expression either in in vitro studies of neutrophils from healthy individuals or in in vivo studies of patients receiving GM-CSF. Stimulation of unprimed neutrophils with CIKM5 induced a rapid transient increase in intracellular calcium levels to 181% of resting levels. However, incubation with GM-CSF did not further augment the calcium transients above the stimulated level. The mechanism by which GM-CSF induces an enhanced respiratory burst in response to cross-linking of Fc gamma RII remains to be elucidated, but is not related to receptor expression or increases in receptor mediated calcium mobilization.     

Role of N-terminal domain of histidine-rich glycoprotein in modulation of macrophage Fc gamma receptor-mediated phagocytosis.

A brief exposure of murine peritoneal inflammatory macrophages to plasma histidine-rich glycoprotein (HRG; 77,000-81,000 MW) for 1-2 hr increased Fc gamma receptor (Fc gamma R) expression and phagocytic function in these cells. However, a continual culture of the cells without the presence of HRG for the next 18-48 hr resulted in down-regulation of Fc gamma R expression and phagocytic function. Similarly, HRG decreased Fc gamma RII expression in less differentiated human THP-1 monocytic cells during treatment for 18 hr, as determined by cellular ELISA and metabolic labelling. The molecular mechanism by which HRG regulates Fc gamma R expression is unknown. However, at a relatively high concentration (> 1 microgram/ml), HRG altered the cellular metabolism by increasing cellular protein synthesis but reducing protein secretion. These observations suggest a likely mechanism for the HRG-mediated reduction of Fc gamma R expression. A degraded HRG (40,000 MW) which possessed an identical N-terminal sequence as that of the native HRG was capable of decreasing macrophage Fc gamma R expression and phagocytosis. The results indicate that the functional domain of HRG responsible for binding to macrophages is localized to the N-terminal half.     

Differential regulation by leukotrienes and calcium of Fc gamma receptor-induced phagocytosis and Syk activation in dendritic cells versus macrophages

Macrophage (M?) phagocytosis via the Fc receptor for immunoglobulin G (Fc gammaR) requires the spleen tyrosine kinase (Syk) and serves an important antimicrobial function. We have reported previously that Fc gammaR-mediated ingestion and Syk activation in M? are amplified by and depend on the proinflammatory lipid mediator leukotriene B4 (LTB4). Although Fc gammaR-mediated ingestion is also important for antigen uptake, there is no information about LTB4 regulation of these processes in dendritic cells (DCs). In this study, we compared murine bone marrow (BM)-derived DCs to M? from BM, peritoneum, and the pulmonary alveolar space. Neither phagocytosis nor Syk activation in DCs was influenced by exogenous LTB4. Unlike the various M? populations, Syk activation in DCs was likewise unaffected by pharmacologic or genetic strategies to inhibit endogenous LTB4 synthesis or to block the high-affinity LTB4 receptor BLT1. DCs were refractory to regulation by LTB4 despite the fact that they expressed BLT1 and mobilized intracellular calcium in response to its ligation. This resistance to LTB4 in DCs instead reflected the fact that in contrast to M?, Syk activation in DCs was itself entirely independent of calcium. These results identify a fundamental difference in Fc gammaR signaling between DCs and M?, which may relate to the divergent, functional consequences of target ingestion in the two cell types.     

Distinct intracytoplasmic sequences are required for endocytosis and phagocytosis via murine Fc{gamma}RII in mast cells

In the present work, we studied the phagocytic and endocyticproperties of murine FcRII in mast cells. Mouse mast cells expresshigh-affinity receptors for monomeric IgE and three low-affinityreceptors for complexed IgG: FcRIIb1, FcRIIb2, and FcRIII. Inprevious studies we showed that, when aggregated by multivalentligands, murine FCRIII, but not FcRII, triggers the releaseof inflammatory mediators and cytokines by mast cells. UponFcR aggregation, mast cells not only release intracellular materials,they also internalize particulate and soluble immune complexes.We compared the ability of the two FcRII isoforms to triggerphagocytosis and endocytosis in RBL-2H3 cells stably transfectedwith cDNAs encoding wild-type, deleted, and tyrosine mutantFcRIIb1 or FcRIIb2. We found that FcRIIb2, but not FcRIIb1,triggered both phagocytosis and endocytosis. We identified distinctintracytoplasmic sequences necessary for FcRIIb2-mediated endocytosisand phagocytosis respectively, and we observed that two tyrosineresidues, located in each of these sequences, are critical forendocytosis and/or phagocytosis. Our data indicate that thetwo internalization pathways diverge as early as signal transductlon.     

Modulation of human peripheral blood lymphocyte Fc gamma receptors by immune complexes: recovery of Fc gamma receptors in the presence of normal human serum.

When normal human peripheral blood lymphocytes (PBL) were incubated at 37 degrees with soluble transferrin anti-transferrin (TAT) complexes a significant reduction in the proportion of PBL bearing receptors for the reacted Fc portion of IgG(Fc gamma R) was found. Following incubation of such complex-treated PBL in normal human serum the proportion of Fc gamma R + PBL, as assessed by rosette formation with chicken erythrocytes (E) presensitized with rabbit antibody (A) was found to be significantly increased. Such serum-mediated recovery of Fc gamma R was not affected by pretreating PBL with cycloheximide. Recovery was found to be species restricted, Ca++ dependent and confined to a serum fraction containing molecules of relatively low molecular weight (less than 90,000 Mr). Following absorption with EA the restorative capacity of human serum was lost. These findings suggest that following modulation of human PBL-Fc gamma R by immune complexes, receptors may be restored to the cell surface from a serum pool of 'fluid-phase' Fc gamma R. The origin and biological significance of serum Fc gamma R is not known but it is conceivable that they play an important role in immunoregulation.     

Regulation of the expression of Fc gamma receptor on circulating neutrophils and monocytes in Kawasaki disease.

To investigate the regulation of Fc gamma receptor (Fc gamma R) expression on circulating phagocytes in Kawasaki disease (KD), we analysed the expressions of Fc gamma RI, II and III on neutrophils and monocytes in 20 patients with KD, 10 with a bacterial infection (BI), 10 with a viral infection (VI), and 10 healthy controls (HC) using flow cytometric analysis. The KD patients had a significantly higher level of Fc gamma RI expression on neutrophils, but not on monocytes, than the BI, VI and HC patients. Fc gamma RII expression on neutrophils was significantly higher in KD, BI and VI than HC, but there was no significant difference in Fc gamma RII expression among KD, BI and VI. Fc gamma RIII expression on neutrophils in KD was significantly lower than in VI and HC, but was higher on monocytes. A kinetic analysis of Fc gamma R expression in KD demonstrated the expression of Fc gamma RI and II on neutrophils to decline, but no remarkable change was observed in the monocytes, from the subacute phase through the convalescent phase. In addition, Fc gamma RIII expression on neutrophils increased, while Fc gamma RIII expression on monocytes decreased during the time course of KD. Fc gamma R expression in the acute phase of KD is thus characterized by markedly increased expression of Fc gamma RI on neutrophils, followed by a subsequent decrease, and decreased expression of Fc gamma RIII on neutrophils and increased expression of Fc gamma RIII on monocytes followed by a reverse kinetics during the clinical course. These findings are thus considered to reflect the functional up-regulation of neutrophils and monocytes in KD.     

Fc gamma receptors differ in their structural requirements for interaction with the tyrosine kinase Syk in the initial steps of signaling for phagocytosis

Receptors for the constant region of IgG, Fc gamma receptors, are expressed on the surface of hematopoietic cells, where they mediate signaling events, such as phagocytosis, essential for host defense. Fc gamma receptors also play a role in the pathophysiology of autoimmune diseases. We have demonstrated that members of each of the three classes of human Fc gamma receptors, Fc gamma RI, Fc gamma RII, and Fc gamma RIII, mediate phagocytosis, but that important differences exist in their requirements for phagocytic signaling. For example, the Fc gamma receptors Fc gamma RI and Fc gamma RIIIA induce signaling largely by association with a gamma subunit containing a conserved cytoplasmic motif (ITAM) whose tyrosines are phosphorylated following receptor stimulation. Fc gamma RIIA contains a similar motif in its own cytoplasmic domain and does not require the gamma chain for phagocytic signaling. The tyrosine kinase Syk associates with the cytoplasmic domain of both the Fc gamma receptor gamma chain and Fc gamma RIIA and is required for phagocytosis by both Fc gamma receptor systems. To elucidate the differences in phagocytic signaling by the gamma chain and Fc gamma RIIA, we investigated the requirements for Fc gamma receptor/Syk co-immunoprecipitation, tyrosine phosphorylation, and phagocytosis. Both Fc gamma RIIA and the human gamma chain contain a tyrosine seven amino acids upstream of the ITAM motif. We observed that the upstream tyrosine plays a role in Fc gamma RIIA phagocytic signaling but is not involved in phagocytic signaling by the human gamma chain. Our data also indicate that the two ITAM tyrosines of the human gamma chain and Fc gamma RIIA do not contribute equally to Fc gamma receptor association with Syk kinase and phagocytic signaling. The data indicate that the carboxy-terminal tyrosine of the receptor cytoplasmic domain is especially important both for the interaction with Syk kinase and for phagocytosis. Elucidating such differences in gamma chain and Fc gamma RIIA signaling may be valuable in designing strategies for therapeutic intervention in hematopoietic and immunological disorders.     

alpha 2-macroglobulin stimulation of protein tyrosine phosphorylation in macrophages via the mannose receptor for Fc gamma receptor-mediated phagocytosis activation.

Macrophage phagocytic activity has previously been shown to be increased by binding of modified alpha 2-macroglobulin (alpha 2M) with mannose residues at termini of sugar chains to mannose receptors on macrophages, with a subsequent increase in the number of Fc gamma receptors at the cell surface. In the present study, an examination was made of the association of protein tyrosine kinase with the increase in number of Fc gamma receptors following binding of modified alpha 2M to mannose receptors. The phagocytosis of IgG-opsonized sheep red blood cells through the action of the Fc gamma receptor by modified alpha 2M was inhibited by mannose and herbimycin A and slightly so by genistein. The mannose receptor would thus appear to be associated with tyrosine kinase activity. By Western blotting, tyrosine phosphorylated proteins with molecular weights of 32000, 34000, 36000, 65000, 85000 and 110000 appeared or increased upon treating macrophages with modified alpha 2M. The degree of tyrosine phosphorylated proteins was the same for control macrophages following incubation in the presence of mannose and herbimycin A. Genistein treatment affected only tyrosine phosphorylated proteins of 65000 and 110000. The binding of modified alpha 2M to mannose receptors was demonstrated by the inducement of tyrosine kinase activation that was sensitive to herbimycin A, followed by an increase in Fc gamma receptors and consequently greater phagocytosis.     

The Fc valency of an immune complex is the decisive factor for binding to low-affinity Fc gamma receptors

Tetanus toxoid (TT) was complexed with two human monoclonal antibodies. The antibodies recognized different, nonrepeating epitopes. The complexes formed were characterized by gel filtration and isokinetic sucrose density gradient centrifugation. It was found that in antigenic excess the separate antibodies formed a complex of one antibody molecule and two TT molecules [IgG1-(TT)2 and IgG3-(TT)2]. In cases where equal amounts of TT and both antibodies were mixed, a dimeric complex [IgG1-(TT)2-IgG3] was formed. The binding of these immune complexes to human neutrophils and eosinophils was studied. Whereas the immune complexes containing one antibody did not bind to either cell type, the two-antibody complex bound to both. This indicates that not the sterical change in the Fc part of an antibody molecule after binding an antigen, but the Fc valency of an immune complex is the decisive factor in Fc receptor interaction with neutrophilic and eosinophilic granulocytes.