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.     

Immune elimination of aging platelets by autologous monocytes: role of membrane-specific autoantibody

Membrane-bound IgG was found only on old populations of platelets from normal individuals. This IgG could be dissociated from senescent cells by repeatedly heating the cells. Heat-eluted IgG (He-IgG) prepared from senescent red blood cells was capable of binding to either heat-treated old platelets or Vibrio cholerae neuraminidase (VCN)-treated young platelets, suggesting expression of a common age-dependent antigen on the senescent red blood cells and old platelets. We analyzed the role of membrane-bound IgG in the immune elimination of aging platelets by direct phagocytosis of different platelet subpopulations by autologous monocytes in vitro. While removal of He-IgG from old platelets inhibited their phagocytosis, preincubation of either heat-treated old or VCN-treated young platelets promoted phagocytosis of these cells by autologous monocytes. The phagocytosis of senescent cells required intact IgG on these cells. Either removal of Fc fragments from He-IgG or treatment of autologous monocytes with Fc fragments prior to the phagocytosis assay resulted in a marked reduction of phagocytosis (greater than 75%). We conclude that Fc receptors on the monocytes and the presence of membrane-specific IgG are crucial elements for immune elimination of senescent platelets.     

Quantitative assessment of Fc receptor expression and function during in vitro differentiation of human monocytes to macrophages.

The expression and function of IgG Fc receptors on peripheral blood monocytes and monocytes differentiating in vitro, within hydrophobic membranes, to macrophages have been determined. Three characteristic functional stages could be discerned, and these were reflected in the surface expression of specific IgG binding sites. Stage 1, represented by fresh monocytes, is characterized by efficient binding and phagocytosis of IgG-coated particles, although uptake was limited by the cell size. Within 1 day of culture, monocytes transform into Stage 2 cells. These bind and ingest particles with high IgG surface density only and these functions are exquisitely sensitive to inhibition by IgG in solution. The functional loss is paralleled by a decrease of the number of specific IgG binding sites, despite a gradual increase in cell size. Between Day 4 and Day 8, macrophages acquired the capacity to bind and ingest particles opsonized to a low degree, their phagocytic capacity is strongly enhanced, the number of IgG binding sites increases by a factor greater than 10, despite a moderate increase in cell size only. Mature human macrophages have about six times the Fc receptor number of blood monocytes, but the average binding affinity is decreased. It appears that the phagocytic capacity is related to the number of IgG binding sites, and the susceptibility to inhibition by IgG in solution is related to their surface density.     

Fcgamma receptor-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes in vitro

Although convincing evidence exists for the role of immunoglobulin G (IgG) antibodies in immunity to malaria, antibody titres do not usually predict protection. In this study we have assessed the interaction between Plasmodium falciparum-infected erythrocytes (PE), opsonized with immune serum containing different amounts of IgG antibody isotypes, with either THP-1 cells, ex-vivo human monocytes or IIAI.6 transfectant cells expressing Fc(gamma)RIIa-Arg/Arg131 or -His/His131 allotypes. Our results show that PMA-treated THP-1 cells were capable of phagocytosing serum-opsonized PE by Fc(gamma)RI (CD64) and Fc(gamma)RIIa (CD32), acting synergistically. The known Fc(gamma)RIIa polymorphism motivated us to examine its influence on IgG isotype-mediated phagocytosis of opsonized PE with human monocytes and the IIAI.6 transfectant cells expressing either allelic forms. Regardless of the cell type, PE phagocytosis with Fc(gamma)RIIa-His/His131 was highest following opsonization with a predominantly IgG3-containing immune serum pool. In contrast, PE phagocytosis with Fc(gamma)RIIa-Arg/Arg131 tended to be higher with an IgG1-containing pool. These results suggest a genetically determined influence of effector cell phenotype on IgG antibody-pathogen interaction in P. falciparum malaria.     

Gammaglobulin for intravenous use induces an Fc gamma receptor-specific decrement in phagocytosis by blood monocytes

Intravenous gammaglobulin (IVIG) increases the circulating platelet count in patients with idiopathic thrombocytopenic purpura in association with mononuclear phagocyte system blockade and decreased blood monocyte phagocytosis. To investigate whether IVIG treatment induces an Fc receptor-specific defect or a generalized phagocytic dysfunction which might impair host defenses, we studied the influence of IVIG on Fc receptor-dependent and Fc receptor-independent internalization by monocytes. In purified monocytes incubated in suspension with IVIG (10 mg IgG/ml) for 48 hr, our data demonstrate that Fc receptor-mediated phagocytosis of IgG-sensitized erythrocytes (EA) is significantly decreased. By contrast, there was no significant change in the phagocytosis of neuraminidase-treated erythrocytes which are internalized via the B-glucan receptor. A similar decrease in EA ingestion was observed in adherent monocytes incubated with IVIG. By contrast, the capacity of IVIG-treated adherent monocytes to internalize tannic acid-treated erythrocytes, an Fc receptor-independent probe, was the same as that of control cells. These findings demonstrate that IVIG does not induce a generalized phagocytic blockade, but rather a selective deficit in Fc gamma receptor-mediated internalization. Thus, Fc receptor-independent mechanisms may provide adequate mononuclear phagocyte system function for host defense in patients receiving IVIG.     

The functional activity of human monocytes passively sensitized with monoclonal anti-D suggests a novel role for Fc gamma RI in the immune destruction of blood cells.

The role of Fc gamma RI in the immune destruction of blood cells is uncertain as serum IgG levels are sufficient to competitively inhibit interactions between this high-affinity receptor and sensitized red cells. In the current study, it is proposed that, rather than functioning as a receptor for opsonized red cells, Fc gamma RI might, under appropriate conditions, mediate the passive sensitization (or 'arming') of human macrophages with IgG antibodies resulting in the in vivo destruction of unsensitized cells expressing the corresponding antigen. To examine this hypothesis, Fc gamma RI-bearing human monocytes and U937 cells were first passively sensitized by incubation in vitro with human monoclonal anti-D, and then incubated with D-positive red cells. The uptake of monoclonal anti-D by U937 cells was rapid and, in the presence of 2.5 micrograms/ml IgG1 or IgG3 anti-D, was almost complete after 5 min at 37 degrees. Subsequent incubation of passively sensitized U937 cells in an IgG-free medium for 1 hr at 37 degrees resulted in the loss from the cell surface of approximately 50% cell-bound IgG; the remaining cell-bound IgG was lost more slowly despite repeated washing. In functional assays, passively sensitized monocytes (M-IgG) mediated adherent, phagocytic and chemiluminescent (CL) responses to D-positive red cells. After incubation of M-IgG in 50% v/v fresh normal human serum (FNHS) for 2 hr, sufficient anti-D remained bound to monocytes to promote the adherence of red cells. The adherence and phagocytosis of red cells by M-IgG was enhanced by the simultaneous addition of 50% FNHS, probably owing to the binding of low levels of C3bi to red cells. In contrast, phagocytic and CL responses of unsensitized monocytes to anti-D-sensitized red cells (E-IgG) were abrogated in the presence of 0.25% v/v FNHS, presumably owing to blocking of Fc gamma RI by IgG. It is considered that in vivo, Fc gamma RI may mediate the passive sensitization of macrophages in close proximity with antibody-secreting cells in the reticular network of the splenic cords. Once 'armed' in this way, macrophages may destroy cells expressing the appropriate antigen.     

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.     

Effect of polymeric IgG on human monocyte functions

Fc and iC3b receptors are involved in various biological functions of phagocytic cells, such as immune adherence and phagocytosis of opsonized particles, degranulation and superoxide generation. In the present study we examined the expression of specific receptors for the Fc portion of IgG (FcR) and for iC3b (CR3), a cleavage product of the third complement component, on human monocytes following in vitro treatment with polymeric and monomeric IgG. Interaction of polymeric IgG (fluid phase) with the monocyte membrane led to a concomitant modulation of both Fc and iC3b receptors. Monomeric IgG, however, down modulated Fc receptor expression only if surface bound. Under these conditions, no concomitant modulation of the iC3b receptor could be observed. The down modulation of Fc and iC3b receptors induced by fluid-phase IgG polymers was also accompanied by a decrease in monocyte functions as expressed by reduced Fc receptor-mediated phagocytosis, decreased release of oxygen metabolites following stimulation by aggregated IgG and opsonized zymosan, as well as in impaired killing of bacteria. These data suggest that a down modulation of Fc and iC3b receptors might have important implications for host defense mechanisms, since interaction with these receptors is required for the proper elimination of many pathogens.     

Receptor specific probes for the study of Fc gamma receptor specific function.

Human leukocytes express three distinct families of receptors for the Fc region of IgG (Fc gamma R). We have prepared erythrocytes (E) coated with monoclonal anti-Fc gamma R antibodies for the study of receptor specific phagocytosis using biotin and streptavidin. In this technique, both the E and the monoclonal antibody (mAb) are biotinylated and coupling of the mAb to the E occurs through the use of streptavidin. The same biotin/streptavidin principle was used to prepare E coated with human IgG. Using this technique, receptor specific probes or probes coated with natural ligand (IgG) can be prepared rapidly with the use of small amounts of mAb or IgG. Finally, we have used these receptor specific probes to demonstrate that all three families of Fc gamma R (Fc gamma RI, Fc gamma RII and Fc gamma RIII) expressed on human monocytes and human macrophages are phagocytic receptors.     

Affinity labeling of the Fc receptor on human monocytes using bifunctional cross-linking agents

To affinity label the Fc receptor on human monocytes, Fc fragments of monoclonal human IgG1 radiolabeled with iodine 125 were covalently bound to the surface of intact monocytes using a variety of bifunctional cross-linking agents including ethylene glycol bis(succinimidyl succinate), dithio-bis-(succinimidyl proprionate), maleimidobenzoyl N-hydroxysuccinimide, glutaraldehyde and dimethyl suberimidate. After cross-linking, cells were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography. Each of these cross-linkers caused a portion of cell-bound Fc fragments to form a covalent complex with a monocyte membrane component. This complex migrated on electrophoresis with an apparent molecular weight of 120,000. Deducting the molecular weight of Fc fragments alone (53,000) the molecular weight of the second component of the complex therefore was about 67,000. A similar estimate of receptor size also was obtained after reduction with dithiothreitol. Complex formation was potently inhibited by unlabeled Fc fragments, IgG1 or IgG3, all of which would be expected to compete with Fc fragments for IgG Fc receptor on human monocytes, but was not inhibited by Fab fragments, IgG2 or IgG4, which do not bind avidly to this receptor. By quantitating the amount of complex formed in the presence of varying concentrations of labeled ligand, it could be demonstrated that complex formation was saturable, and that Fc fragments formed complexes with avidity comparable to that with which Fc fragments bound to receptors on intact monocytes. The findings establish the feasibility of using radiolabeled Fc fragments to affinity label the IgG Fc receptors on human leukocytes. Potential advantages of this approach to studying receptor structure are discussed.     

Regulation of macrophage Fc receptor expression and phagocytosis by histidine-rich glycoprotein.

Regulation of macrophage Fc receptor (Fc gamma R)-mediated phagocytic function by histidine-rich glycoprotein (HRG) was investigated. Pretreatment of oil-elicited inflammatory mouse peritoneal macrophages with HRG for 1-3 hr increased their Fc gamma R-mediated binding and phagocytosis of IgG-opsonized sheep erythrocyte conjugates (EA). A significant reduction of Fc gamma R-dependent EA binding and phagocytosis occurred after pretreatment of macrophages with HRG for more than 8 hr. These results indicate that HRG is capable of modulating Fc gamma R expression in a biphasic fashion, which directly affects the overall efficiency of phagocytosis. HRG differentially regulated the functions of Fc gamma R subclasses. For example, HRG reduced the efficiency of Fc gamma RII (Fc gamma 2b/gamma 1R)-dependent phagocytosis of erythrocytes conjugated with monoclonal IgG2b or IgG1 by macrophages pretreated with HRG for 24 hr. However, when similar studies were performed using erythrocytes coated with monoclonal IgG2a, HRG was less effective in inhibiting Fc gamma RI (Fc gamma 2aR)-dependent phagocytosis. As an HRG-binding glycosaminoglycan, heparin failed to block the regulatory function of HRG on macrophages. Similarly, interferon-gamma (IFN-gamma) was not capable of blocking the functional activity of HRG. These studies suggest that HRG regulates macrophage function via a novel pathway different from that of heparin or IFN-gamma.     

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.     

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 isotype of autoantibodies influences the phagocytosis of antibody-coated platelets in autoimmune thrombocytopenic purpura

Autoimmune thrombocytopenic purpura (AITP) is an acquired autoimmune bleeding disorder, characterized by isolated thrombocytopenia because of destruction of auto‐antibody‐coated platelets by Fc‐receptor‐mediated phagocytosis. The destruction of autoantibody‐sensitized platelets by FcγR‐bearing phagocytic cells and the following antigen presentation are considered to play a key role for the pathophysiology of AITP. Although different isotypes of AITP‐mediating autoantibodies, e.g. IgG, IgM and IgA, are frequently found in AITP patients, their role in the pathophysiology of AITP remains unclear. Using a flow cytometric monocyte‐based phagocytosis assay, we investigated the impact of disease‐associated autoantibody isotype in antibody‐mediated phagocytosis of platelets. Platelets, labelled with 5‐chloromethyl fluorescein diacetate (CMFDA), were incubated with AITP patients’ serum characterized by pure IgG or IgM antiplatelet autoantibodies. Labelled platelets were incubated with monocytes. Phagocytosis was defined as the product of percentage of CMFDA‐positive monocytes and mean fluorescence intensity of CMFDA. Adherence of platelets to monocytes was quantified by anti‐CD61‐PerCp in a CMFDA⁺ CD14⁺ gate. IgG‐coated platelets showed a significantly higher phagocytic index than IgM‐coated platelets (mean 796 ± 157 versus 539 ± 78, P < 0.01). There were no significant differences regarding platelet adherence to monocytes. The isotype of autoantibodies influences the quantity of in vitro phagocytosis of autologous platelets by monocytes. Therefore, the AITP‐mediating autoantibody isotype should be considered more carefully in pathophysiologic models and furthermore in diagnostic, therapeutic and prognostic approaches in AITP.     

Antibody-mediated erythrolysis and erythrophagocytosis by human monocytes, macrophages and activated macrophages. Evidence for distinction between involvement of high-affinity and low-affinity receptors for IgG by using different erythroid target cells.

Antibody-dependent cellular cytotoxicity (ADCC) and Fc receptor-mediated phagocytosis were determined with human monocytes, monocyte-derived macrophages and activated macrophages, using rabbit IgG-covered sheep red blood cells (EAs) and anti-D-treated human erythrocytes (EAhu) as target cells. Monocyte and macrophage-mediated ADCC were distinguished by different kinetics, monocytes lysing either target more rapidly than macrophages. Macrophage activation by recombinant IFN-gamma (rIFN-gamma) led to a marked increase in ADCC activity against EAhu. This manifested in increased lysis of optimally sensitized target cells, in a sustained lysis of target cells carrying low antibody densities, and as an enhanced resistance to lysis inhibition by competing fluid-phase inhibition by competing fluid-phase IgG. All these effects were less striking when EAs were the target cells. Phagocytosis of EAs by rIFN-gamma-treated cells was strongly suppressed, regardless of the amount of antibody on the target cells, and susceptibility to inhibition by fluid-phase IgG was slightly increased. By comparison, phagocytosis of EAhu was depressed to a lesser degree, and susceptibility to inhibition by fluid-phase IgG was reduced when macrophages were rIFN-gamma treated. These and other experiments suggested that the functional triggering of monocytes and macrophages by EAs involved, at least in part, low-affinity Fc receptors (FcR), whereas EAhu interacted with macrophages via high-affinity FcR. It is shown elsewhere that rIFN-gamma treatment of macrophages increases the expression of high-affinity FcR, but not low-affinity FcR (Jungi, Lerch & Brcic, 1987). Differences in the rIFN-gamma-induced functional alterations assessed with EAhu or with EAs are interpreted therefore as being a consequence of differential involvement of high-affinity FcR and of low-affinity FcR in mediating an effector function. For monitoring rIFN-gamma-induced alterations in the effector capacity EAs are more appropriate targets since up-regulation of high-affinity FcR has a smaller influence on the response to this type of target. Using metabolic inhibitors, ADCC could be dissociated from ingestion suggesting that ADCC is not a post-phagocytic event.     

Distinctive role of IgG1 and IgG3 isotypes in Fc gamma R-mediated functions

Polyclonal and monoclonal anti-Rh (D) antibodies of IgG1 and IgG3 subclass were evaluated for their capacity to sensitize erythrocytes and (i) to trigger monocyte and K-cell mediated antibody-dependent cellular cytotoxicity (ADCC); (ii) to mediate binding to monocyte and lymphocyte Fc gamma R; (iii) to stimulate phagocytosis by monocytes. All antibodies were equally effective in mediating monocyte or activated U937 cell ADCC but IgG1 was more active than IgG3 in K-cell mediated ADCC. IgG3-sensitized erythrocytes inhibited IgG1-induced lysis, suggesting that each subclass engages the same Fc gamma R receptor but that lysis requires a further 'signal' that the IgG3 molecule can not deliver. Two monoclonal IgG3 anti-D antibodies were shown to have higher binding (two times) and phagocytic (three times) indices than IgG1 antibody for monocytes; similar differences were observed for polyclonal IgG1 and IgG3 antibodies. The same pattern was observed in an EA rosette assay when a total lymphocyte population was used; however, this difference was not seen with a B-cell depleted (T+ null cell) lymphocyte population.     

Binding sites for C-reactive protein on human monocytes are distinct from IgG Fc receptors

Previous investigations have provided evidence to suggest that C-reactive protein (CRP), an acute-phase reactant, binds to human monocytes at a membrane site that is either identical to or physically associated with IgG Fc receptors. To characterize further the relationship between monocyte CRP binding sites and IgG Fc receptors, monocytes were allowed to attach to surfaces coated with IgG or CRP and binding-site redistribution was assessed. Binding was measured by using protein-coated sheep erythrocytes (E). When attached to control (gelatin or albumin) surfaces, greater than 60% and 43% of monocytes formed rosettes with E-IgG and E-CRP, respectively. Following adherence to surface immobilized CRP, the proportion of cells binding E-IgG was unchanged; however, fewer than 20% of monocytes bound E-CRP. When attached to IgG-coated surfaces, fewer than 20% of monocytes formed rosettes with either E-IgG or E-CRP. In order to determine whether the unidirectional modulation of CRP and IgG binding sites was the result of CRP binding directly to a subclass of IgG Fc receptors, fluid-phase IgG-blocking studies were performed. When monocyte monolayers were preincubated with either monomeric or heat-aggregated IgG, a dose-dependent reduction in E-IgG binding was observed. In contrast, all concentrations of fluid-phase IgG failed to inhibit monocyte binding of E-CRP. These data indicate that CRP binds to human monocytes at a site physically associated with but distinct from IgG Fc receptors.     

The type of interaction with Fc gamma R in human monocytes determines the efficiency of the generation of oxidative burst.

Receptors for the Fc fragment of IgG (Fc gamma R) have a well-documented role in the generation of oxidative burst. It is tempting to speculate that the type of interaction with Fc gamma R could be a mechanism of regulation of this process. Here we report on a comparative study of the induction of oxidative burst in human monocytes activated by means of different types of interaction with Fc gamma R. We studied non-primed monocytes obtained by centrifugal elutriation from healthy donors. These cells were submitted to Fc gamma R interactions following two distinct models: one, using particulate material (IgG-SRBC leading to phagocytosis or rosetting), and another using soluble reagents followed by cross-linking of the receptors (monoclonal antibodies against Fc gamma RI and Fc gamma RII and natural ligands, namely several isotypes of murine and human IgG). Phagocytosis and oxidative burst were studied simultaneously in the monocytes, following the methodology described recently. Human non-primed monocytes were able to generate a very obvious oxidative burst response after activation of Fc gamma R by particulate material. The same response was observed when Fc gamma RII was blocked by monoclonal antibodies. Ingestion was not necessary for activation of the oxidative burst, since the model of rosetting induced a level of burst generation similar to the one obtained in the phagocytic process. Cross-linking of Fc gamma RI by soluble reagents induced production of reactive oxidative intermediates (ROI) only when the ligand-binding site of the receptor was involved. These data lead to the conclusion that Fc gamma R interaction with soluble or particulate material induces oxidative burst in non-primed human monocytes only when the binding site of natural ligands is involved. The type of interaction also determines the efficiency of the generation of ROI. This fact could represent a regulatory mechanism.     

Interferon gamma-treated human macrophages display enhanced cytolysis and generation of reactive oxygen metabolites but reduced ingestion upon Fc receptor triggering

Human monocyte-derived macrophages treated with recombinant IFN-gamma (rIFN-gamma) and control cells were assessed for three distinct effector functions, all mediated by Fc receptors. rIFN-gamma-primed macrophage displayed markedly reduced phagocytosis of IgG antibody-coated erythrocytes. In contrast, antibody-dependent cytotoxicity towards IgG-antibody-coated erythrocytes and IgG-antibody-coated erythrocyte-induced generation of reactive oxygen metabolite production were increased. The decreased phagocytosis was observed microscopically, as well as in a spectrometric and a radiometric phagocytosis assay. Evidence is presented that the observed impairment in phagocytosis is not the result of increased extracellular lysis or intracellular catabolism of IgG-antibody-coated erythrocytes and that it is not observed with particles ingested in an Fc receptor-independent manner. Enhanced production of reactive oxygen metabolites was detected most clearly by measurement of luminol-dependent chemiluminescence. Antibody-dependent cellular cytotoxicity was shown to proceed also under conditions impeding phagocytosis, and rIFN-gamma-treated macrophage exerted enhanced antibody-dependent cellular cytotoxicity under these conditions too. In all three assays, functional alterations were optimally expressed after a treatment with 500 U/ml for 46 hr. Analysis at the single-cell level revealed that the IFN-gamma-induced alterations were expressed by all macrophages and not the property of distinct macrophage subpopulations. This and earlier studies suggest that the modulation of Fc receptor-mediated macrophage effector functions by IFN-gamma is in part a post-receptor-binding event.     

The interaction of ruminant IgG with receptor type II for IgG on human phagocytes

The interaction of ruminant IgG with human phagocytes was assessed using Fc receptor (FcR)-mediated ingestion and the triggering of a respiratory burst as effector functions indicative of receptor-specific interaction. In monomeric form, ruminant IgG was three to five orders of magnitude less potent than homologous IgG in inhibiting FcR-specific phagocytosis by monocytes. However, when attached to tanned sheep erythrocytes (Es-T), ruminant IgG was opsonic, as it promoted enhanced phagocytosis of Es-T, comparable to ingestion of rabbit IgG-coated Es. This phagocytosis was inhibitable by high concentrations of human IgG in the fluid phase. Moreover, Es-T precoated with ferritin could be opsonized to a similar degree by anti-ferritin IgG from rabbit and cow. However, only bovine IgG1, but not IgG2, was opsonic. Bovine and goat IgG of some, but not other, suppliers were inactive. Similar results were obtained by measuring the respiratory burst triggered by heat-aggregated IgG, using a luminol-enhanced chemiluminescence assay. Thus, human IgG and ruminant IgG stimulated monocytes and, to a lesser extent, polymorphonuclear leucocytes (PMN), to generate CL. Depending on the manufacturer, some preparations of bovine and goat IgG were inactive, and bovine IgG2 failed to induce CL. These findings prove that certain ruminant IgG preparations, including bovine IgG1 interacting weakly with homologous PMN and monocytes, do interact with human PMN, monocytes and macrophages in a FcR-specific manner when offered in complexed form. Inhibition studies suggest that bovine IgG1 interacts mainly with human FcR type II. In contrast, bovine IgG2, regarded as cytophilic for homologous PMN, fails to interact with human PMN, monocytes and macrophages.