Human Amnion as a Model for IgG Transport

ABSTRACT: The ability of fresh human amnion to bind and internalize horseradish peroxidase-Iabeled IgG (IgG-HRP) was examined in an in vitro Ussing chamber system. The amnion demonstrated unique cell membrane receptors for the Fc portion of IgG molecules (FeyR). The FcγR exhibit exquisite specificity and affinity for IgG monomers as demonstrated by staining with labeled IgG. Labeled IgA, IgM, F(ab')2 fragments of IgG, aggregated IgG, and antigen-antibody complexes all failed to bind to the amnionic epithelial cells. Binding was only minimally affected by changes in ionic strength or pH when viewed at the light microscopic level. The FcγR are located on both the apical and basal cell membranes. The binding of IgG-HRP to the amnion cell membrane was detectable within 1 min, and internalization of the ligand occurred within 5 min. No binding of IgG-HRP was observed following treatment of the membrane with 0.25% trypsin for 30 min at room temperature. Incubation of the amnion at 4°C or in the presence of colchicine or cytochalasin D prevented internalization of the IgG-HRP. These experiments demonstrate FcγR on human amnionic epithelial cells that both bind and internalize IgG, thus allowing the amnion to be used as a model system for studying IgG transport.     

Chimeric Fc receptors identify immunoglobulin-binding regions in human FcγRII and FcϵRI

FcγRII and Fc?RI are functionally distinct cell surface receptors for immunoglobulin (Ig); FcγRII binds IgG with low affinity, whereas Fc?RI binds IgE with high affinity, yet they are homologous in structure and sequence having extracellular regions containing two Ig-like domains with 38% amino acid identity. Chimeric receptors derived from human FcγRII and FcγRI were produced by exchanging homologous regions of the two receptors to define binding region(s) for IgG in FcγRII and IgE in Fc?RI. Firstly, a chimeric form of the Fc?RI α chain was produced by replacing the transmembrane region and cytoplasmic tail with that of FcγRII. This mutant α chain could be expressed on the cell surface independently of associated β and γ subunits, and retained high-affinity IgE binding, indicating that the extracellular region of the FcγRI α chain is sufficient for high-affinity IgE binding. Secondly, to identify the role of the individual domains in Fc binding of both FcγRII and FcγRI, chimeric receptors were generated by exchanging the first extracellular domains between FcγRII and the α chain mutant and used to demonstrate that the second extracellular domain of both receptors contains region(s) directly involved in Ig binding. Additional chimeric receptors were constructed to localize the Ig interactive regions in domain two of FcγRII and FcγRI; these identified a single region of IgG binding in FcγRII located between residues Ser¹³⁶ to Val¹⁶⁹, and at least three independent IgE binding regions in the FcγRI α chain, between residues Trp⁸⁷ to Lys¹²⁸, Tyr¹²⁹ to Asp¹⁴⁵, and Ser¹⁴⁶ to Val¹⁶⁹.     

The IgG and Transferrin Receptors of the Human Syncytiotrophoblast Microvillus Plasma Membrane*

ABSTRACT: Fcγ receptor activity has been demonstrated using a radioisotopic assay on human syncytiotrophoblast microvillus plasma membrane (StMPM) vesicle preparations isolated from normal full-term placentas. Prior chaotropic extraction to remove extrinsic membrane protein from StMPM preparations was shown to increase radiolabeled IgG uptake by up to 30%. Polyacrylamide disc gel electrophoresis (PAGE) has been used to indicate that the StMPM receptor for transferrin solubilizes in 1% sodium deoxycholate and retains its binding activity for transferrin. Quantitative data for both Fcγ and transferrin receptors obtained following study of isolated StMPM preparations are summarized. Current concepts for the potential role of trophoblast Fey and transferrin receptors in the mechanisms of transplacental passage of IgG and iron, respectively, from mother to fetus are illustrated and discussed.     

Membrane-Spanning Fcγ Receptor III Isoform Expressed on Human Placental Trophoblasts

PROBLEM: Fc receptor for immunoglobulin (FcγR) is an important mediator of immunological functions in the feto-maternal relationship. We have demonstrated by immunohistochemical means that three distinct classes of FcγRS are expressed in the different cell components of the human placenta. METHOD: In this study, FcγRIII isoform expressed on placental trophoblasts (PTs) was investigated by indirect immunofluorescence and cDNA cloning. PTs, isolated from human term placenta by digestion with proteolytic enzyme, were reacted with monoclonal antibodies (MAb) against the Fc-γRs and other surface markers of leukocytes and subjected to flow cytometric analysis. RESULTS: PTs were positively stained with 3G8 and Leul lb against FcγRIII, partially stained with MAb against MHC class I, but not with 32.2 (FcγRI), IV3 (FcγRII), or MAbs against CD4, CD19, or CD56, indicating that only low affinity receptor, FC7RIII, is γexpressed on PTs. The DNA sequence of cloned FcγRIII CDNA from PTs by PCR was identical to that of natural killer (NK) cell isoform, including the position of the stop codon that differs from the granulocyte isoform by several nucleotide substitutions. We further analyzed the susceptibility of PTs against phosphatidylinositol specific phospholipase C (PI-PLC) to determine the structural topology of PT isoform. While the reactivity with 3G8 on PTs was not influenced by treatment with PI-PLC, that on granulocytes was significantly diminished with PI-PLC. CONCLUSIONS: This result confirmed that FcγRIII on PTs is a membrane-spanning molecule, and that it is distinctive from PI anchoring FcγRIII on granulocytes.     

Selection of a human anti-RhD monoclonal antibody for therapeutic use: impact of IgG glycosylation on activating and inhibitory Fc gamma R functions

The substitution of plasmatic anti-RhD polyclonal antibodies by a monoclonal antibody (mAb) for preventing the hemolytic disease of the newborn (HDN) is an important issue due to supply and safety concerns. Since it has been suggested that FcgammaR are involved in the prevention of HDN, the in vitro functional properties of two anti-RhD mAbs differing through their glycosylation profiles were compared using FcgammaR-based assays to select a candidate mAb. T125(YB2/0), a low fucosylated antibody, bound strongly to both activating FcgammaRIII and inhibitory FcgammaRII, as opposed to its highly fucosylated counterpart. It also exerted a strong ADCC against RhD+ RBCs and a potent FcgammaRIIB-mediated inhibition of cytokine release. Moreover, an in vivo RhD+ red blood cells (RBCs) clearance assay showed that this antibody exhibits a RhD+ RBCs clearance as potent as polyclonal anti-RhD antibodies in NOD-SCID mice. Thus, T125(YB2/O) has been selected to be tested for the prevention of anti-RhD allo-immunization.     

Kinetic Analysis of the Interaction between Recombinant Human FcRIα and Serum IgEs from Allergic Patients

Binding of IgE to the high-affinity IgE receptor (FcεRI) is the essential event for allergic reaction. Although there are many reports on binding kinetics between myeloma IgE and FcεRI, little is known about the kinetics between heterogeneous polyclonal IgE in the serum and FcεRIα. To elucidate the binding characteristics of heterogeneous serum IgE, we measured kinetic parameters of binding between IgE from allergic patients and a recombinant ectodomain of the human FcεRIα subunit by real-time interaction analysis based on surface plasmon resonance. Purified IgE monomer from the plasma of allergic patients displayed kinetics for the interaction with FcεRIα similar to those of myeloma IgE. In the case of crude IgE samples from allergic patients, one of seven specimens showed significantly higher affinity than highly purified IgE, suggesting that it is possible for IgEs in this specimen to form complexes of higher molecular weight.     

Regulation of Fcγ receptors and immunoglobulin G-mediated phagocytosis in mouse microglia

As resident macrophages in the CNS, microglia can transform from a surveillance state to an activated phenotype in response to brain injury. During this transition microglia become highly capable phagocytic cells. Invading pathogens undergo opsonization with immunoglobulins and microglia recognize these opsonized pathogens through interaction with their cognate F_c receptors. In mice, both FcγRI and FcγRIIb receptors are involved in IgG-mediated phagocytosis of opsonzied pathogens. At sites of inflammation, microglial activity is regulated by T-cell derived cytokines. Here we first investigated the effects of IFN-γ, IL-4, IL-13 and GM-CSF on expression of FcγRI and FcγRIIb mRNA levels in both primary microglia and microglial cell line N9. Using quantitative real-time PCR we show that IFN-γ induced a 4-fold increase in the mRNA level of FcγRI but did not induce changes in FcγRIIb expression. IL-4 and IL-13 induced approximately 2-fold increases in expression of FcγRIIb mRNA, but had no effect on FcγRI expression. GM-CSF increased both FcγRI and FcγRIIb mRNA expression. We then characterized the ability of these same cytokines to regulate phagocytosis of immune complexes composed of IgG and the bacteria Staphylococcus aureus. IFN-γ and GM-CSF both induced approximately 2-fold increases in IgG-mediated phagocytosis whereas IL-4 and IL-13 both decreased IgG-mediated phagocytosis by about one-third. None of the cytokines influenced basal levels of phagocytosis. These findings demonstrate a highly selective cytokine-induced regulation of both phagocytosis-related Fcγ receptor subtypes and IgG-mediated phagocytosis itself in microglia. This selective regulation has implications for our understanding of the pathophysiology of CNS infection and autoimmune disease.     

Expression and Identification of Immunological Activities of the HIV‐gp120N‐Human Interferon Gamma Fusion Protein

The human immunodeficiency virus type 1 (HIV‐1) envelope glycoprotein gp120 is a vaccine immunogen that has been studied extensively. To enhance the immune response of cells against HIV‐1 gp120, we tested the coexpression of gp120N with interferon‐γ (IFN‐γ) as an immune adjuvant. Two recombinant prokaryotic plasmids were constructed: the pET44b‐HIV‐1‐gp120N plasmid construct carried the HIV‐1 gp120N gene (pET44‐gp120N), whereas the pET44b‐HIV‐1‐gp120N‐IFN‐γ plasmid construct carried a fusion gp120N‐IFN‐γ gene (pET44b‐gp120N‐IFN‐γ). Target protein expression was achieved in E. coli BL21 (DE3) cells by chemical induction. To test the immunological activity of the proteins, mice were injected with a control, gp120N, or the fusion gp120N‐IFN‐γ protein. The serum and spleen cells of the mice were collected for immunological detection. Results showed that specific T lymphocyte proliferation and the expression of the Th1‐type cytokines (IL‐2 and IFN‐γ) were higher in the gp120N‐IFN‐γ group than the other two groups (P < 0.05). No difference was observed in the expression levels of the Th2‐type cytokines (IL‐4 and IL‐10; P > 0.05). These results suggest that IFN‐γ plays a prominent role as an immune adjuvant when coexpressed with HIV‐1 gp120N. IFN‐γ enhances the specific cell immune response of mice against HIV‐1 gp120. Anat Rec, 292:381–386, 2009. © 2009 Wiley‐Liss, Inc.     

Chlamydophila pneumoniae Infection of Human Aortic Endothelial Cells Induces the Expression of FC γ Receptor II (FcγRII)

Chronic endothelial infection is believed to be one of the factors able to cause endothelial cell damage and trigger the onset of human atherosclerosis. Chlamydophila pneumoniae infects endothelial cells and has received special attention because of both epidemiological and experimental evidence supporting its role as a risk factor for atherosclerosis. It is also possible that otherwise independent risk factors for atherosclerosis may have synergistic effects. Immune phenomena, such as the formation of circulating immune complexes (IC) containing modified LDL and corresponding antibodies, have been linked to the development of coronary artery disease. The antibodies involved in the immune response to modified lipoproteins are predominantly of the pro-inflammatory IgG1 and IgG3 subclasses. However, it is difficult to understand how circulating IC could cause endothelial damage and initiate the atherosclerotic process, unless they were formed in the subendothelial space or immobilized by endothelial cells. The last hypothesis would be possible if endothelial cells expressed Fcγ receptors. Healthy endothelial cells do not express Fcγ receptors, but endothelial cells infected by a variety of infectious agents do. Thus we decided to investigate whether infection of endothelial cells with C. pneumoniae is also able to cause the expression of Fcγ receptors. The expression of Fcγ receptors (CD64, 32, and 16) on human aortic endothelial cells infected with C. pneumoniae for 4, 24, 36, and 48 h was studied by flow cytometry. Twenty-four hours after infection 30–40% of the endothelial cells had detectable inclusion bodies, 8–9% of the total number of cells (approximately 25% of the infected cells) expressed FcγRII, and about 1.5–2% (5% of infected cells) expressed FcγRI and FcγRIII. Double-staining studies confirmed that the expression of FcγRII was limited to C. pneumoniae-infected endothelial cells. We conclude that C. pneumoniae infection induces primarily the expression of FcγRII by endothelial cells and this may be a significant link between two proposed pathogenic mechanisms involved in the pathogenesis of human atherosclerosis.     

Disease activity in systemic lupus erythematosus is associated with an altered expression of low-affinity Fcγ receptors and costimulatory molecules on dendritic cells

Dendritic cells (DCs) play a pivotal role in the interface between immunity and maintenance of peripheral tolerance. The capture of immunoglobulin G (IgG)-containing immune complexes (ICs) by low-affinity Fcγ receptors (FcγRs) expressed on DCs may influence the immunogenicity/tolerogenicity of these cells, depending on the activating/inhibitory potential of FcγRs. Because of the key role that low-affinity FcγRs play in determining the magnitude of the response in IC-driven inflammation, these receptors are likely to play a role in autoimmune diseases, such as systemic lupus erythematosus (SLE). To evaluate if an altered expression of costimulatory molecules and/or FcγRs could account for disease severity, we evaluated the expression of these molecules on immature and mature DCs derived from peripheral blood monocytes of SLE patients and healthy donors. Our results show an increased expression of the costimulatory molecules CD40 and CD86. Furthermore, the ratio of CD86/CD80 is higher in SLE patients compared with healthy donors. Conversely, while the expression of activating FcγRs was higher on DCs from SLE patients, expression of inhibitory FcγRs was lower, compared with DCs obtained from healthy donors. As a result, the activating to inhibitory FcγR ratio was significantly higher in DCs from SLE patients. The altered ratio of activating/inhibitory FcγRs on mature DCs showed a significant correlation with the activity of SLE, as determined by the SLE Disease Activity Index (SLEDAI) score. We postulate that the increased ratio of activating/inhibitory FcγRs expressed on DCs from SLE patients can contribute to the failure of peripheral tolerance in the IC-mediated phase of autoimmune pathogenesis.     

Divergent effects of FcγRIIIA ligands on the functional activities of human natural killer cells in vitro

The Fcγ receptor (R)IIIA (CD16) plays an important role in regulating the cytotoxic and non-cytotoxic functions of human natural killer (NK) cells. Some anti-CD 16 monoclonal antibodies (mAb) have been shown to stimulate NK activity, while human monomeric (m) IgG induces dose-dependent inhibition of NK activity. To explore further these interactions mediated via FcγRIIIA, purified NK cells were cultured for 2–3 days in the presence of mIgG, 3G8 mAb, interleukin-2 (IL-2) or a combination of mIgG or 3G8 with IL-2. Binding of mIgG or 3G8 to FcγRIIIA induced divergent effects of functions of cultured NK cells: 3G8 mAb + IL-2 induced dose-dependent inhibition of proliferation attributable to apoptosis; in contrast, mIgG + IL-2 significantly increased NK cell proliferation. Incubation of NK cells in the presence of mIgG up-regulated expression of surface activation markers (CD69, IL-2Rα, ICAM-1), cytotoxicity, cytokine production (IL-1β, IFN-γ and TNF-α) and release of soluble IL-2R. Thus, mIgG binding to FcγRIIIA induced stimulatory signals in human NK cells, leading to up-regulation of IL-2Rα expression, cell proliferation and cytokine release.     

Human Fcγ receptors compete for TGN1412 binding that determines the antibody's effector function

The first‐in‐human clinical trial of the CD28‐specific monoclonal antibody (mAb) TGN1412 resulted in a life‐threatening cytokine release syndrome. Although TGN1412 was designed as IgG4, known for weak Fc:Fcγ receptor (FcγR) interactions, these interactions contributed to TGN1412‐induced T‐cell activation. Using cell lines (TFs) expressing human FcγRI, ‐IIa, ‐IIb, or ‐III, we show that TGN1412 and TGN1412 as IgG1 and IgG2 are bound by FcγRs as it can be deduced from literature. However, upon coculture of TGN1412‐decorated T cells with TFs or human primary blood cells, we observed that binding capacities by FcγRs do not correlate with the strength of the mediated effector function. FcγRIIa and FcγRIIb, showing no or very minor binding to TGN1412, mediated strongest T cell proliferation, while high‐affinity FcγRI, exhibiting strong TGN1412 binding, mediated hardly any T‐cell proliferation. These findings are of biological relevance because we show that FcγRI binds TGN1412, thus prevents binding to FcγRIIa or FcγRIIb, and consequently disables T‐cell proliferation. In line with this, FcγRI^?FcγRII⁺ but not FcγRI⁺FcγRII⁺ monocytes mediate TGN1412‐induced T‐cell proliferation. Collectively, by using TGN1412 as example, our results indicate that binding of monomeric IgG subclasses does not predict the FcγR‐mediated effector function, which has major implications for the design of therapeutic mAbs.     

Mechanisms of Apoptosis of T-Cells in Human Tuberculosis

The role of TGF-β TNF-α FasL and Bcl-2 in apoptosis of CD4 T-cells during active TB was studied. Coculture of PBMC from TB patients with neutralizing antibodies to TGF-β or TNF-α decreased spontaneous (P ≤ 0.05) and MTB-induced (P≤ 0.02) T-cell apoptosis by 50–90%, but effects were not additive. Interestingly, only levels of TGF-β in supernatants correlated with rates of spontaneous and MTB-induced apoptosis. FasL surface and mRNA expression were higher in unstimulated and MTB-stimulated PBMC from patients than controls, and neutralization of FasL abrogated apoptosis of T-cells from patients only. Intracellular Bcl-2 protein was lower among unstimulated CD4 T-cells from patients than those from controls (P ≤ 0.02), and MTB stimulation reduced intracellular Bcl-2 content in CD4 T-cells from patients only (P ≤ 0.001). These findings may indicate that, during TB, predisposition of CD4 T-cells to apoptosis may involve both low expression of Bcl-2, and excessive expression of TGF-β TNF-α and FasL.     

An IgG-transporting Fc receptor expressed in the syncytiotrophoblast of human placenta

During normal human pregnancy, maternal IgG crosses the placenta and provides passive immunity for the fetus. In so doing, IgG passes through two cellular barriers: the syncytiotrophoblast and the fetal capillary endothelium. The Fc region of IgG is required for its transport across the placenta, but the Fc receptors responsible have not been identified definitively. We recently reported the isolation from a placental cDNA library of clones encoding the α chain of a human homologue of the major histocompatibility complex class I-related Fc receptor, the neonatal Fc receptor (FcRn). In mice, FcRn is essential for the transport of maternal IgG to the fetus and the neonate. We report here the localization of human FcRn mRNA within the placenta by in situ hybridization, and of human FcRn protein by immunohistochemistry. Both methods show that human FcRn is expressed in syncytiotrophoblast, and is, thus, appropriately located to transport maternal IgG across the first barrier. We confirm previous findings that specific binding of IgG to placental membranes is greater at pH 6.0 than pH 7.5. This corresponds with the pH dependence of IgG binding to FcRn and is consistent with the presence of FcRn in syncytiotrophoblast. We propose a transport model in which maternal IgG binds FcRn at low pH in endosomes within the syncytiotrophoblast. FcRn is not expressed in fetal capillary endothelia, and the mechanism of IgG transport across the second barrier remains unknown.