Effector functions of a monoclonal aglycosylated mouse IgG2a: Binding and activation of complement component C1 and interaction with human monocyte Fc receptor

Myasthenia gravis (MG) is an autoimmune disease in which immunoglobulin G (IgG) antibodies (Abs) bind to acetylcholine receptors (AChR) or to functionally related molecules in the postsynaptic membrane at the neuromuscular junction. IgG crystallizable fragment (Fc)-mediated effector functions, such as antibody-dependent complement deposition, contribute to disease development and progression. Despite progress in understanding Ab-mediated disease mechanisms, immunotherapy of MG remained rather unspecific with corticosteroids and maintenance with immunosuppressants as first choice drugs for most patients. More specific therapeutic IgG Fc-based platforms that reduce serum half-life or effector functions of pathogenic MG-related Abs are currently being developed, tested in clinical trials or have recently been successfully translated into the clinic. In this review, we illustrate mechanisms of action and clinical efficacies of emerging Fc-mediated therapeutics such as neonatal Fc receptor (FcRn)-targeting agents. Furthermore, we evaluate prospects of therapies targeting classical Fc receptors that have shown promising therapeutic efficacy in other antibody-mediated conditions. Increased availability of Fc- and Fc receptor-targeting biologics might foster the development of personalized immunotherapies with the potential to induce sustained disease remission in patients with MG.

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Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

mBio ◽

10.1128/mbio.01624-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 64

Author(s):

Caitlin E. Mullarkey ◽

Mark J. Bailey ◽

Diana A. Golubeva ◽

Gene S. Tan ◽

Raffael Nachbagauer ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Fc Receptor ◽

Igg Antibodies ◽

Specific Antibodies ◽

Effector Functions ◽

Iga Antibodies ◽

Specific Igg ◽

Optimal Protection ◽

Specific Igg Antibodies

ABSTRACTBroadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protectionin vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using anin vitroassay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.IMPORTANCEThe present study provides evidence that broadly neutralizing HA stalk-specific antibodies induce downstream Fc-mediated neutrophil effector functions. In addition to their ability to neutralize, this class of antibodies has been shown to rely on Fc-Fc receptor interactions for optimal protectionin vivo. Curiously, neutralizing antibodies that bind the HA head domain do not require such interactions. Our findings build on these previous observations and provide a more complete picture of the relationship between stalk-specific antibodies and cells of the innate immune compartment. Furthermore, our data suggest that the ability of HA stalk-specific antibodies to mediate Fc-Fc receptor engagement is epitope dependent. Overall, this work will inform the rational design of improved influenza virus vaccines and therapeutics.

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Characterization of the human monocyte high affinity Fc receptor (hu FcRI)

Molecular Immunology ◽

10.1016/0161-5890(88)90015-6 ◽

1988 ◽

Vol 25 (3) ◽

pp. 243-250 ◽

Cited By ~ 26

Author(s):

Gary Peltz ◽

Katherine Frederick ◽

Clark L. Anderson ◽

B.Matija Peterlin

Keyword(s):

Human Monocyte ◽

Fc Receptor ◽

High Affinity

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Lipopolysaccharide Regulates Macrophage Fluid Phase Pinocytosis Via CD14-Dependent and CD14-Independent Pathways

Blood ◽

10.1182/blood.v93.11.4011.411a29_4011_4018 ◽

1999 ◽

Vol 93 (11) ◽

pp. 4011-4018

Author(s):

Maikel P. Peppelenbosch ◽

Marjory DeSmedt ◽

Tessa ten Hove ◽

Sander J.H. van Deventer ◽

Johan Grooten

Keyword(s):

Septic Shock ◽

Biological Effects ◽

Fluid Phase ◽

Cell Types ◽

Fc Receptor ◽

Effector Functions ◽

Receptor Mediated Endocytosis ◽

Monocytes And Macrophages ◽

Dual Action ◽

Stimulation Of

Lipopolysaccharide (LPS) is a mediator of inflammation and septic shock during bacterial infection. Although monocytes and macrophages are highly responsive to LPS, the biological effects of LPS in these cell types are only partially understood. We decided, therefore, to investigate the influence of LPS on macrophage pinocytosis and Fc receptor–mediated endocytosis, two prominent and related macrophage effector functions. We observed that LPS did not greatly influence endocytosis in either macrophages or monocytes, but did exert a dual action on pinocytosis: at lower concentrations (0.1 to 100 ng/mL), LPS caused a decrease in pinocytosis in both macrophages and monocytes, whereas at higher LPS concentrations, enhanced pinocytosis in macrophages was observed. Detoxified LPS was two orders of magnitude less potent in producing these effects. After inhibition of the LPS receptor CD14, the LPS-induced decrease in pinocytosis was absent, and stimulation of pinocytosis at lower LPS concentrations was unmasked. We conclude that LPS can influence pinocytosis via CD14-dependent and CD14-independent signaling pathways. Furthermore, as addition of LPS to macrophages effected pinocytosis but not Fc receptor–mediated endocytosis, these two processes are independently regulated in macrophages.

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Detection of human monocyte-reactive alloantibodies by flow cytometry after selective downmodulation of the Fc receptor I

Blood ◽

10.1182/blood.v78.8.2150.2150 ◽

1991 ◽

Vol 78 (8) ◽

pp. 2150-2156 ◽

Cited By ~ 9

Author(s):

RW Kuijpers ◽

MC Dooren ◽

AE von dem Borne ◽

WH Ouwehand

Keyword(s):

Polyclonal Antibodies ◽

Hla Class I ◽

Human Monocyte ◽

Fc Receptor ◽

Antigenic Determinants ◽

Flow Cytofluorometry ◽

Human Sera ◽

Antigen System ◽

Murine Monoclonal Antibodies ◽

Reproducible Manner

Abstract Monocyte-reactive human alloantibodies may be of importance in situations such as transfusion reactions and bone marrow and kidney transplantation. So far, only complement-binding monocyte-reactive antibodies can be detected with a cytotoxicity assay. No antiglobulin assays are yet available that also detect noncomplement-fixing monocyte- reactive antibodies. The binding of monomeric IgG with high affinity to the Fc receptor I (FcRI) on monocytes has severely hampered the development of such an assay until now. We report on the selective removal of the FcRI from monocytes to test human sera in a flow cytofluorometry assay for the presence of monocyte-reactive IgG alloantibodies. Selective downmodulation of FcRI was accomplished by incubating the cells with murine monoclonal antibodies against FcRI followed by a second incubation with goat-antimouse IgG polyclonal antibodies. With such modified cells, human complement-binding and noncomplement-binding IgG and IgM alloantibodies against polymorphic determinants of the HLA class I and II glycoproteins, the human monocyte antigen system and polymorphic antigenic determinants of the LFA complex, can be detected in a sensitive and reproducible manner.

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Transmembrane signaling: an ion-flux-independent model for signal transduction by complexed Fc receptors.

The Journal of Cell Biology ◽

10.1083/jcb.99.6.2231 ◽

1984 ◽

Vol 99 (6) ◽

pp. 2231-2240 ◽

Cited By ~ 12

Author(s):

L C Pfefferkorn

Keyword(s):

Signal Transduction ◽

Fc Receptor ◽

Ion Flux ◽

Transmembrane Signaling ◽

Myristate Acetate ◽

Acridine Orange Staining ◽

Effector Functions ◽

Opsonized Zymosan ◽

Pre Treatment ◽

Independent Model

Fluxes of Na+/K+ that precede effector functions in stimulated phagocytes are thought to play a role in signal transduction. To examine this hypothesis, phagocytosis, phagosomal acidification, and superoxide anion generation (O2-) were stimulated in media in which the Na+ was replaced with K+ or choline+. Counts of particles internalized and assessment of acidification of the phagosomes by acridine orange staining indicated that Na+/K+ fluxes were not necessary for phagocytosis or phagosomal acidification in J774.2 macrophages. Phagocytosis mediated by the ionophoretic Fc receptor gamma 2b/gamma 1 of J774.2 macrophages was equally independent of a Na+ gradient. Na+/K+ fluxes did not dictate the rate of O2- generation in human monocytes. Therefore, in at least these three effector functions, Na+/K+ fluxes stimulated by Fc- and non-specific receptor binding play neither a signaling nor an enhancing role. An ion-flux-independent model for transmembrane signaling by the Fc receptor is proposed. Others have shown that there is an apparent dependence on the external Na+ concentration for O2- generation and lysosomal secretion by neutrophils. These neutrophils had been pre-treated with NH4+ during a routine purification step. O2- generation stimulated by opsonized zymosan or phorbol myristate acetate, by monocytes or monocyte-derived macrophages, and phagocytosis of opsonized zymosan by J774.2 macrophages, showed dependence on external Na+ only if these cells had been pre-treated with NH4+. Brief NH4+ pre-treatment would be expected to acidify the cytoplasm of the cells. The reversal of this acidification is known to require Na+ for H+ extrusion through the Na+/H+ antiport, thus explaining the apparent Na+ dependence.

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Fc-Receptor Interactions Regulate Both Cytotoxic and Immunomodulatory Therapeutic Antibody Effector Functions

Cancer Immunology Research ◽

10.1158/2326-6066.cir-15-0120 ◽

2015 ◽

Vol 3 (7) ◽

pp. 704-713 ◽

Cited By ~ 64

Author(s):

David J. DiLillo ◽

Jeffrey V. Ravetch

Keyword(s):

Fc Receptor ◽

Therapeutic Antibody ◽

Effector Functions ◽

Receptor Interactions

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Comparable modulation of human monocyte functions by commercial factor VIII concentrates of varying purity

Blood ◽

10.1182/blood.v71.6.1662.bloodjournal7161662 ◽

1988 ◽

Vol 71 (6) ◽

pp. 1662-1668

Author(s):

JW Mannhalter ◽

R Ahmad ◽

H Leibl ◽

J Gottlicher ◽

HM Wolf ◽

...

Keyword(s):

Factor Viii ◽

Immune Modulation ◽

General Feature ◽

Human Monocyte ◽

Fc Receptor ◽

Human Monocytes ◽

Receptor Assay ◽

Factor Viii Concentrates ◽

Blood Group Substances ◽

Concentrate Preparation

Our previous observation on immune modulation induced by a given factor VIII (F VIII) concentrate preparation was extended by showing that the immune-modulating capacity is a more general feature of F VIII products and is independent of product purity. Interaction of human monocytes with therapeutic concentrations of various F VIII concentrates (0.2 to 2 IU F VIII/mL, six different F VIII concentrates from four manufacturers) led to a significant reduction in the expression of IgG Fc receptors in the membrane of these cells (F VIII concentrate-induced downmodulation of the receptor). This Fc receptor downmodulation was achieved by a short (1-hour) incubation of human monocytes with F VIII concentrates 16 hours prior to the Fc receptor assay and did not correlate with the respective product's IgG content. Although the IgG concentrations of the different products varied greatly (from 1.0 to 177.3 mg/1,000 IU F VIII), all products behaved comparably with respect to Fc receptor downmodulation (F VIII-treated monocytes: 34% +/- 7% to 44% +/- 4% rosette-forming cells; controls in the absence of F VIII: 83% +/- 5%). Furthermore, we also were able to demonstrate that heat treatment of F VIII, now used by virtually every manufacturer to eliminate contaminating viruses, had no effect on the respective products' Fc receptor-modulating capacity. The immune-modulating component was characterized as being a high-molecular-range compound containing IgG, IgM, F VIII, and blood group substances (most likely a combination of immune complexes and immunoglobulin aggregates). This compound is present in comparable amounts in both high-purity and intermediate-purity products and apparently copurifies with F VIII during the manufacturing process.

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Detection of human monocyte-reactive alloantibodies by flow cytometry after selective downmodulation of the Fc receptor I

Blood ◽

10.1182/blood.v78.8.2150.bloodjournal7882150 ◽

1991 ◽

Vol 78 (8) ◽

pp. 2150-2156

Author(s):

RW Kuijpers ◽

MC Dooren ◽

AE von dem Borne ◽

WH Ouwehand

Keyword(s):

Polyclonal Antibodies ◽

Hla Class I ◽

Human Monocyte ◽

Fc Receptor ◽

Antigenic Determinants ◽

Flow Cytofluorometry ◽

Human Sera ◽

Antigen System ◽

Murine Monoclonal Antibodies ◽

Reproducible Manner

Monocyte-reactive human alloantibodies may be of importance in situations such as transfusion reactions and bone marrow and kidney transplantation. So far, only complement-binding monocyte-reactive antibodies can be detected with a cytotoxicity assay. No antiglobulin assays are yet available that also detect noncomplement-fixing monocyte- reactive antibodies. The binding of monomeric IgG with high affinity to the Fc receptor I (FcRI) on monocytes has severely hampered the development of such an assay until now. We report on the selective removal of the FcRI from monocytes to test human sera in a flow cytofluorometry assay for the presence of monocyte-reactive IgG alloantibodies. Selective downmodulation of FcRI was accomplished by incubating the cells with murine monoclonal antibodies against FcRI followed by a second incubation with goat-antimouse IgG polyclonal antibodies. With such modified cells, human complement-binding and noncomplement-binding IgG and IgM alloantibodies against polymorphic determinants of the HLA class I and II glycoproteins, the human monocyte antigen system and polymorphic antigenic determinants of the LFA complex, can be detected in a sensitive and reproducible manner.

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