Fc receptors in immune thrombocytopenias: a target for immunomodulation?

In autoimmune disease, Fc receptors (FcRs) form the interface between immune effector cells and their antibody-coated targets, and as such are attractive targets for immunomodulatory therapy. In this issue of the JCI, two highly novel studies of Fc–FcR interactions provide new insights into the role of FcRs in immune thrombocytopenia. Asahi et al. utilized a comprehensive platform of immunological assays to examine the mechanism underlying Helicobacter pylori–associated immune thrombocytopenic purpura, and Ghevaert et al. describe a specially designed antibody that saturates binding sites on fetal platelets without initiating FcγR-mediated platelet phagocytosis, preventing the binding of pathological maternal anti-HLA antibodies that cause fetomaternal alloimmune thrombocytopenia (see the related articles beginning on pages 2939 and 2929, respectively). These reports illustrate how a remarkably detailed molecular understanding of the FcR network may translate into new therapeutic strategies with high clinical impact.

The interactions between immune cells and their target cells in autoimmune diseases have been the focus of much attention, and intense efforts have been made to manipulate the signaling pathways involved. The great majority of studies have examined T and B cells, and recently there has been increased interest in the role of Tregs as deft orchestrators of the immune response (1, 2). In contrast, macrophages have largely been investigated for their ability to execute intracellular killing and are considered to be the mobile but passive “clean-up men” of the host defense system. Part of their weaponry, which only devotees care to distinguish into subgroups, comprises the Fc receptors (FcRs). Seminal studies, initially from Ravetch’s group and subsequently from the Lazarus laboratory, developed key insights into the network of FcRs expressed on macrophages, and the interactions between these phagocytic cells and antibodies emerged as attractive targets for immunomodulatory therapy (3, 4). Two articles in this issue of the JCI involve very different manipulations of the Fc-FcR interaction in order to increase our understanding of the pivotal role played by the FcR network in the pathogenesis of immune thrombocytopenia. Both reports are highly clinically relevant. In the first study, Asahi et al. examined the changes in the balance of FcRs expressed by patients with immune thrombocytopenia purpura (ITP) and Helicobacter pylori infection in order to explore the mechanism of platelet recovery that has been observed in these individuals following treatment to eradicate H. pylori (5). In the second study, Ghevaert et al. report the development and preclinical testing of a recombinant antibody designed to prevent FcR-mediated alloimmune destruction of platelets, which may have potential as a treatment approach for fetomaternal alloimmune thrombocytopenia (FMAIT) (6).

The central immunopathological disturbance in immune thrombocytopenia is the destruction of antibody-coated platelets by phagocytic cells in the reticuloendothelial system (7). Circulating monocytes and resident macrophages in the spleen and liver bind to the exposed Fc portion of platelet-associated IgG molecules via Fc receptors for IgG, namely FcγR. This system first entered the limelight when it became clear that a primary mechanism of action of two first-line therapies for ITP, steroids and i.v. immunoglobulin (IVIG), occurred via interference with FcγR-mediated platelet clearance. The earliest theories for the mechanism of action of IVIG were built on the observation by Fehr et al. in 1982, and ourselves a year later, that treatment with IVIG in non-splenectomized patients with ITP prolonged the clearance of radiolabeled, antibody-coated red blood cells, suggesting competitive inhibition of FcγR-bearing phagocytes in the spleen (8, 9). This development set the stage for the design of more targeted therapies against the FcγR system, with the goal of improving efficacy and avoiding the therapeutic use of human blood products.

Soon thereafter, more specific FcγR-blocking treatments were explored, including i.v. infusions of the immunoglobulin anti-D (which binds specifically to the erythrocyte D antigen) (10) and infusion of a monoclonal anti-FcγRIII antibody (11). Another approach was to modify the IgG in IVIG by digesting the Fc portion in order to change its interaction with the FcγR system. The partially digested product was less effective than intact IVIG in children with ITP (12). However, infusions of isolated Fc fragments of IgG were shown to have similar effects to intact IgG on platelet counts in children with ITP, confirming that Fc-FcγR interactions were important in mediating the therapeutic effects of IVIG (13). These crude manipulations of IgG were abandoned, and among these agents only i.v. anti-D continued forth into routine clinical usage. Nonetheless, these studies illustrated the potential of modulating the interactions between circulating antibodies and FcRs.

Over time, as additional FcRs were identified, the complexity of the FcR system was revealed (Figure 1). As early as 1964, Brambell hypothesized that there existed a process to recycle IgG, later shown to involve the neonatal FcR, FcRn. This FcR is unique among FcRs in that it is a heterodimer consisting of an MHC-1–related glycoprotein bound to a β2 microglobulin protein and has been studied as a therapeutic target to prevent the recycling of autoantibodies in autoimmune disease and, in doing so, shorten autoantibody half-lives (14, 15). Importantly, FcRn also mediates transplacental passage of maternal IgG into the fetus (16, 17). Subsequent exploration of the FcγR system resulted in the remarkable discovery of distinct inhibitory and activating FcγRs; in particular, description of the inhibitory receptor FcγRIIB initiated a new era in studies of FcR manipulation (18, 19).

Figure 1

The activating and inhibitory human FcγRs. Humans have one inhibitory FcγR, FcγRIIB, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) as its intracellular signaling domain. Upon binding to Fc fragments, the ITIM recruits negative regulatory signaling proteins. Fc binding to the other FcγRs, including FcγRIIA, induces recruitment of proteins that are involved in activation signaling, via immunoreceptor tyrosine-based activation motifs (ITAMs), which typically consist of a ligand-binding α-chain. FcγRI and FcγRIIIA have signal-transducing γ-chain dimers (indicated by SS). As reported by Asahi et al. in this issue of the JCI (5), the balance between the numbers of inhibitory (FcγRIIB) versus activating (FcγRIIA) FcγRs is disturbed in patients with ITP and H. pylori infection, with downregulation of the inhibitory receptor FcγRIIB. Eradication of H. pylori was found to normalize the FcγR balance and reduce opsonophagocytosis of platelets by macrophages of the reticuloendothelial system. There is one high-affinity receptor, FcγRI. The other FcγRs have low to medium affinity for the Fc portion. Unlike the other transmembrane receptors, FcγRIII is a glycosylphosphatidylinositol-linked protein.

The realization that the in vivo action of an IgG antibody binding to an FcγR-bearing cell depended on the net balance of activating versus inhibitory FcγR signaling led to the next major clinically related breakthrough, which provided insight into the mechanisms underlying the effect of IVIG in the treatment of ITP. First, Samuelsson et al. reported that the protective effect of IVIG in an anti-platelet antibody–mediated murine model of ITP was dependent on the presence of FcγRIIB and that IVIG administration increased the expression of this inhibitory receptor by splenic macrophages (3). These findings have since been confirmed and dramatically extended. An elegant series of preclinical studies by Lazarus’s group (20) examined the downstream signaling pathways of FcγRs, and the results indicated that the therapeutic effect of IVIG in antibody-mediated murine ITP resulted from its interaction with DCs, in that DCs pre-incubated with IVIG in vitro could recapitulate the therapeutic effect of IVIG. These IVIG-primed leukocytes only took effect when the recipient mouse expressed FcγRIIB, although FcγRIIB was not required on the “initiator” DCs, indicating that FcγRIIB was not the direct target of IVIG but a critical downstream mediator (20). Furthermore, the ability of DCs to ameliorate ITP was maintained in immunodeficient mice lacking T and B cells, suggesting that DCs do not merely act via modulation of antibody production by B cells or of the T cell compartment, but directly interact with phagocytes of the innate immune system to prevent destruction of opsonized platelets. The findings of Samuelsson et al. (3) and the subsequent studies demonstrated the clinical importance of this hitherto unrecognized central role for FcγRIIB and set the stage for the current study by Asahi and colleagues of the mechanism of the effect of H. pylori to exacerbate and/or perpetuate ITP (5).

It is generally accepted that the presence of H. pylori infection may contribute to the pathogenesis and persistence of immune thrombocytopenia in patients with ITP and that eradication of the organism can result in an increase in platelet count in a substantial fraction of infected patients. Despite this clinical wisdom, the underlying mechanism of the effect of H. pylori to cause thrombocytopenia has remained unclear. Suggested hypotheses have included molecular mimicry of H. pylori antigens by platelet/megakaryocyte glycoproteins and infection-related perturbation of the immunoregulatory system, thereby promoting the production of autoreactive antibodies (21). The platform of immunological assays employed by Asahi et al. in their current study (5) suggests that H. pylori causes or exacerbates ITP by downregulating FcγRIIB and that eradicating H. pylori restores the balance by shifting toward the inhibitory FcγR phenotype with less active opsonophagocytosis, thereby ameliorating the immune-mediated platelet destruction.

The very high degree of homology in the extracellular domains of FcγRIIA and FcγRIIB has been an almost insurmountable obstacle to previous studies of these receptor subtypes (22). In their current study, Asahi et al. (5) employed cell permeabilization in order to use intracellular antibodies specific to the C-terminal portion of FcγRIIB for flow cytometric assays, in combination with mRNA analysis to determine the FcγRIIA/FcγRIIB balance. Future investigation utilizing the very recently developed specific discriminatory antibodies may allow finer delineation of receptor expression and an expanded understanding of the factors that alter the expression of FcγRIIB. Support that the changes in the FcγRIIA/FcγRIIB balance are clinically relevant comes from the temporal correlation of the changes in FcR subtype expression with changes in both phagocytosis assays and the increase in platelet numbers in responders. While this report does not explain why only a portion of H. pylori–infected ITP patients will benefit from H. pylori eradication in terms of their ITP, it provides sound evidence that an imbalance in FcγR signaling may be the most important clinical mechanism underlying H. pylori–associated ITP. We believe this to be the first time that an infection has been shown to alter the natural balance of activating and inhibitory FcRs, with the eradication of the infection being the means to restore the FcR balance.

The report by Ghevaert et al. (6) in this issue of the Journal exploits the FcγR pathway by a totally different approach from that of Asahi et al. (5), this time with the aim of treating FMAIT. FMAIT results from transplacental transfer of maternal antibodies that develop in response to alloimmunization against paternal human platelet antigens (HPAs) expressed on fetal platelets. The HPA-1a antigen is responsible for the great majority of cases of severe FMAIT in individuals of mixed European descent (23). FMAIT is uncommon (affecting approximately 1 in 1,000 births), but it is the most important cause of severe fetal/neonatal thrombocytopenia and is associated with substantial morbidity and mortality due to intracranial hemorrhage, the risk of which is higher if a previous sibling was similarly affected (24). Currently, there is no routine screening for this condition during pregnancy, and antenatal management of siblings of fetuses affected by FMAIT relies on administration of large quantities of IVIG to the mother (1–2 g/kg/wk) with varying degrees of invasive intrauterine monitoring and occasional intrauterine infusions of HPA-compatible platelets.

In contrast to the studies by Asahi et al. (5) on infection-related thrombocytopenia and mechanism of treatment effect, the approach of Ghevaert et al. (6) involves manipulation of the Fc portion of IgG in order to change its interaction with FcRs. Why develop such a complicated treatment for FMAIT? FMAIT presents a more complicated immunopathology than ITP by virtue of involving not only the immunobiology of the mother, fetus, and placenta but also the pregnancy-associated changes to the maternal immune system. Therefore, the challenge faced by Ghevaert et al. was to design an antibody that would bind with high affinity to HPA-1a on platelets, not initiate FcγR-mediated immune clearance of these platelets, and yet have an intact Fc fragment able to interact with FcRn and thereby be transferred via the placenta to the fetus (Figure 2). Previous preclinical studies by this group support the achievement of these aims.

Figure 2

Interfering with FcγR-mediated platelet phagocytosis in FMAIT. In FMAIT, maternal anti-platelet antibodies are transferred across the placenta by the neonatal Fc receptor (FcRn) and mediate clearance of fetal platelets by FcγR-bearing phagocytes (macrophages) in the reticuloendothelial system. In the model proposed by Ghevaert et al. in their current study in this issue of the JCI (6), administration of their newly designed antibody B2G1Δnab would saturate available HPA-1a–binding sites on platelets but not activate FcγR signaling, thereby preventing platelet destruction characteristic of FMAIT.

Ghevaert et al. (6) engineered a specific, non-FcγR–activating antibody construct (termed B2G1Δnab) that saturates available antigenic sites and blocks binding of the pathological maternal antibodies to fetal platelets. The need for preserved interaction with FcRn prevented the use of either single-chain antibodies or of deglycosylating the Fc fragment of the antibody, and of other modifications such as the crude digestions of IgG described above, which would prevent activating interaction with FcγRs. Therefore, the construct was engineered using IgG subclass 2 and 4 residues substituted into an IgG1 backbone (25). IgG2 and IgG4 are known not to activate complement and have a 20- to 100-fold lower affinity for FcγR than IgG1 and IgG3. Using a dually perfused isolated human placental model, they confirmed that transplacental transfer via FcRn was intact (26).

In 19 of 20 maternal sera tested in vitro, up to 95% inhibition of anti–HPA-1a–binding to platelets was achieved using the B2G1Δnab antibody (6). Murine studies confirmed that this recombinant antibody abrogated FcγR-mediated antibody-coated platelet clearance, encouraging further exploration of the feasibility of this approach in patients.

While teasing out the exact mechanism of IVIG therapy in FMAIT has proved difficult, a leading hypothesis is that high-dose weekly infusions of IVIG administered to the mother may block FcRn-mediated transplacental transport of the anti–HPA-1a antibody. This treatment is relatively effective (but very expensive, especially if initiated at 12 weeks of gestation); involves the infusion of large quantities of salt, protein, and water; and is a human blood product. In contrast, there is the enticing possibility that infusion of this synthetic monoclonal antibody B2G1Δnab (6) would only need to be given to the mother, albeit weekly, and therefore, direct intrauterine delivery could potentially be avoided. Extensive in vivo studies of this potential approach will be required to confirm its feasibility.

The race to design effective biological therapies for use in autoimmune thrombocytopenias that have a similar or improved efficacy over IVIG without having the disadvantages of involving human blood products has been in process, although without dramatic success thus far. Two monoclonal antibodies to FcγRIII (3G8 and GMA161) have been used in clinical trials for the treatment of ITP with only moderate efficacy (11, 27). A Syk kinase inhibitor that targets signaling pathways downstream of FcγRs, including FcγRIIA and FcγRIIB, has been shown to achieve a platelet response that could be maintained with continued administration in a majority of patients, albeit with some gastrointestinal toxicity (28).

Why have therapies targeted to the Fc-FcR system not been more successful thus far? As the complexities of the system continue to be revealed, perhaps it is becoming clear that the real challenge is to recapitulate the “social networking” or “class action” of native, intact immunoglobulin. As the current reports by Asahi et al. (5) and Ghevaert et al. (6) illustrate, manipulating FcR-mediated phagocytosis in immune thrombocytopenias remains a highly attractive target for the design of immunomodulatory therapies. In addition, disturbances to the relative expression of the inhibitory receptor FcγRIIB may, as is the case for ITP patients simultaneously infected with H. pylori, underlie other causes of thrombocytopenia in ITP.

B. Psaila is a Fulbright Scholar in Cancer Research and a recipient of a Kay Kendall Leukaemia Fund Traveling Fellowship. This work was also partly supported by Dana Hammond Stubgen, the Children’s Cancer and Blood Foundation, and NIH grant U01 HL072186 (to J.B. Bussel).

Address correspondence to: James B. Bussel, Platelet Disorders Center, Division of Pediatric Hematology-Oncology, Weill-Cornell Medical College of Cornell University, 525 East 68th Street, P-695, New York, New York 10021, USA. Phone: (212) 746-3400; Fax: (212) 746-8609; E-mail: jbussel@med.cornell.edu.

Nonstandard abbreviations used: FcR, Fc receptor; FMAIT, fetomaternal alloimmune thrombocytopenia; HPA, human platelet antigen; ITP, immune thrombocytopenia purpura; IVIG, i.v. immunoglobulin.

Conflict of interest: J.B. Bussel has equity ownership and receives research support from Amgen and GlaxoSmithKline. B. Psaila has no conflict of interest to declare.

Reference information: J. Clin. Invest. doi:10.1172/JCI36451.

See the related articles at Helicobacter pylori eradication shifts monocyte Fcγ receptor balance toward inhibitory FcγRIIB in immune thrombocytopenic purpura patients and Developing recombinant HPA-1a–specific antibodies with abrogated Fcγ receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia.

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