Anja Lux

Impact of Immune Complex Size and Glycosylation on IgG Binding to Human FcγRs

IgG molecules are widely used as therapeutic agents either in the form of intact Abs or as Fc fusion proteins. Although efficient binding of the IgG Fc fragment to cellular FcγRs may be essential to achieve a high cytolytic activity, it may be advantageous for other applications to limit or abolish this interaction. Genetic or biochemical approaches have been used to generate these non–FcγR-binding IgG variants. By using soluble versions of FcγRs and monomeric versions of these altered IgG molecules, it was demonstrated that these IgG variants no longer bind to FcγRs. Importantly, however, these assays do not reflect the physiologic interaction of IgG with low-affinity cellular FcγRs occurring in the form of multimeric immune complexes. In this study, we investigated how the size of an immune complex can affect the interaction of normal and various versions of potentially non–FcγR-binding IgG variants with cellular FcγRs. We show that neither the D265A mutation nor EndoS treatment resulting in IgG molecules with only one N-acetylglucosamine and a fucose residue was fully able to abolish the interaction of all IgG subclasses with cellular FcγRs, suggesting that IgG subclass–specific strategies are essential to fully interfere with human FcγR binding.

FcγRIV deletion reveals its central role for IgG2a and IgG2b activity in vivo

Cellular Fcγ receptors are essential for IgG-dependent effector functions in vivo. There is convincing evidence that selective activating Fcγ receptors are responsible for the activity of individual IgG subclasses. Thus, IgG1 activity is absent in FcγRIII-deficient mice, and several studies suggest that the activity of the most potent IgG subclasses, IgG2a and IgG2b, might be dependent on either individual or a combination of activating FcγRs. To study the role of individual activating FcγRs for IgG subclass activity, we generated an FcγRIV-deficient mouse and showed that a variety of IgG2a- and IgG2b-dependent effector functions are impaired in the absence of this activating Fc receptor in models of autoimmunity and antibody-dependent cellular cytotoxicity.

Monocyte Subsets Responsible for Immunoglobulin G-Dependent Effector Functions In Vivo

Immunoglobulin G (IgG) antibodies confer protection against pathogenic microorganisms, serve as therapeutics in tumor therapy, and are involved in destruction of healthy tissues during autoimmune diseases. Understanding the molecular pathways and effector cell types involved in antibody-mediated effector functions is a prerequisite to modulate these activities. In this study we used two independent model systems to identify innate immune effector cells required for IgG activity in vivo. We first defined the precise repertoire of receptors for the IgG Fc fragment (FcγR) on innate immune effector cells in the blood and on tissue-resident macrophage populations. Despite expression of relevant activating FcγRs on various phagocyte populations, our data indicate that the majority of these cell types are dispensable for IgG activity in vivo. In contrast, IgG-dependent effector functions were selectively impaired in animals lacking the CX(3)CR1(hi)Ly6C(lo)CD11c(int) monocyte subset, which expressed the full set of FcγRs required for IgG activity.

Glycosylation of immunoglobulin G determines osteoclast differentiation and bone loss

Immunglobulin G (IgG) sialylation represents a key checkpoint that determines the engagement of pro- or anti-inflammatory Fcγ receptors (FcγR) and the direction of the immune response. Whether IgG sialylation influences osteoclast differentiation and subsequently bone architecture has not been determined yet, but may represent an important link between immune activation and bone loss. Here we demonstrate that desialylated, but not sialylated, immune complexes enhance osteoclastogenesis in vitro and in vivo. Furthermore, we find that the Fc sialylation state of random IgG and specific IgG autoantibodies determines bone architecture in patients with rheumatoid arthritis. In accordance with these findings, mice treated with the sialic acid precursor N-acetylmannosamine (ManNAc), which results in increased IgG sialylation, are less susceptible to inflammatory bone loss. Taken together, our findings provide a novel mechanism by which immune responses influence the human skeleton and an innovative treatment approach to inhibit immune-mediated bone loss.

Inflammatory monocytes and Fcγ receptor IV on osteoclasts are critical for bone destruction during inflammatory arthritis in mice

Destruction of bone tissue by osteoclasts represents a severe pathological phenotype during inflammatory arthritis and results in joint pain and bone malformations. Previous studies have established the essential role of cytokines including TNFα and receptor–ligand interactions, such as the receptor activator of nuclear factor-kappa B–receptor activator of nuclear factor-kappa B ligand interaction for osteoclast formation during joint inflammation. Moreover, autoantibodies contribute to joint inflammation in inflammatory arthritis by triggering cellular fragment crystallizable (Fc)γ receptors (FcγR), resulting in the release of proinflammatory cytokines and chemokines essential for recruitment and activation of innate immune effector cells. In contrast, little is known about the expression pattern and function of different FcγRs during osteoclast differentiation. This would allow osteoclasts to directly interact with autoantibody immune complexes, rather than being influenced indirectly via proinflammatory cytokines released upon immune complex binding to other FcγR-expressing innate immune cells. To address this question, we studied FcγR expression and function on osteoclasts during the steady state and during acute joint inflammation in a model of inflammatory arthritis. Our results suggest that osteoclastogenesis is directly influenced by IgG autoantibody binding to select activating FcγRs on immature osteoclasts, resulting in enhanced osteoclast generation and, ultimately, bone destruction.

Impact of Differential Glycosylation on IgG Activity

Immunoglobulin G (IgG) molecules are glycoproteins with dual functionality. While participating in the destruction of virally infected cells or healthy tissues during autoimmune disease, IgG antibodies are also used as a therapeutic agent to suppress IgG-triggered autoimmune disease and inflammation. Research of recent years has put the IgG-associated sugar moiety in the spotlight for regulating these opposing activities. This review will focus on how certain IgG glycovariants impact different IgG-dependent effector functions and how this knowledge might be used to further improve the therapeutic effectiveness of this class of molecules.

FcγR dependent mechanisms of cytotoxic, agonistic, and neutralizing antibody activities

Given the widespread use of antibodies of the immunoglobulin G (IgG) class as cytotoxic, immunomodulatory, and neutralizing agents in the therapy of malignant, infectious, and autoimmune diseases, understanding the molecular and cellular mechanisms responsible for their therapeutic activity is of major importance. While Fcγ receptors (FcγR) have well-appreciated roles as effectors of cytotoxic IgG activity, it has only recently become clear that the functionality of immunomodulatory and neutralizing IgG preparations also depends on cellular FcγRs. Here, we review current models of IgG activity in infectious and inflammatory settings, and examine the importance of cell type-specific expression of FcγRs in determining functional outcome. We discuss how this knowledge may be used to improve the activity of therapeutic antibody preparations and outline important areas of focus for future research.

Regulation of autoantibody activity by the IL-23-TH17 axis determines the onset of autoimmune disease

The checkpoints and mechanisms that contribute to autoantibody-driven disease are as yet incompletely understood. Here we identified the axis of interleukin 23 (IL-23) and the TH17 subset of helper T cells as a decisive factor that controlled the intrinsic inflammatory activity of autoantibodies and triggered the clinical onset of autoimmune arthritis. By instructing B cells in an IL-22- and IL-21-dependent manner, TH17 cells regulated the expression of β-galactoside α2,6-sialyltransferase 1 in newly differentiating antibody-producing cells and determined the glycosylation profile and activity of immunoglobulin G (IgG) produced by the plasma cells that subsequently emerged. Asymptomatic humans with rheumatoid arthritis (RA)-specific autoantibodies showed identical changes in the activity and glycosylation of autoreactive IgG antibodies before shifting to the inflammatory phase of RA; thus, our results identify an IL-23–TH17 cell–dependent pathway that controls autoantibody activity and unmasks a preexisting breach in immunotolerance.

The role of sialic acid as a modulator of the anti-inflammatory activity of IgG

Immunoglobulin G (IgG) molecules can have two completely opposing activities. They can be very potent pro-inflammatory mediators on the one hand, directing the effector functions of the innate immune system towards infected cells, tumor cells or healthy tissues in the case of autoimmune diseases. On the other hand, a mixture of IgG molecules purified from the blood of ten thousands of healthy donors is used as an anti-inflammatory treatment for many autoimmune diseases since several decades. It has become evident only recently that certain residues in the sugar moiety attached to the IgG constant fragment can dramatically alter the pro- and anti-inflammatory activities of IgG. This review will focus on sialic acid residues as a modulator of the anti-inflammatory activity and provide an overview of situations where serum IgG glycosylation and sialylation is altered and which molecular and cellular pathways may be involved in this immunomodulatory pathway.

The other side of immunoglobulin G: suppressor of inflammation

Immunoglobulin G (IgG) molecules can have two completely opposite functions. On one hand, they induce proinflammatory responses and recruit innate immune effector cells during infection with pathogenic microorganisms or autoimmune disease. On the other hand, intravenous infusion of high doses of pooled IgG molecules from thousands of donors [intravenous IG (IVIG) therapy] represents an efficient anti‐inflammatory treatment for many autoimmune diseases. Whereas our understanding of the mechanism of the proinflammatory activity of IgG is quite advanced, we are only at the very beginning to comprehend how the anti‐inflammatory activity comes about and what cellular and molecular players are involved in this activity. This review will summarize our current knowledge and focus upon the two major models of either IVIG‐mediated competition for IgG‐triggered effector functions or IVIG‐mediated adjustment of cellular activation thresholds used to explain the mechanism of the anti‐inflammatory activity.