A passionate kiss, then run: exocytosis and recycling of IgG by FcRn

The MHC-class-I-like Fcgamma receptor FcRn recycles immunoglobulin (Ig)G from most cells and transports it bi-directionally across epithelial barriers to affect systemic and mucosal immunity. Recent studies have shown that FcRn rescues IgG from intracellular lysosomal degradation by recycling it from the sorting endosome to the cell surface. Most recycling vesicles fuse completely with the plasma membrane in a classical pattern of exocytosis. Similar to the process seen for neurotransmitter release at synaptic junctions, other vesicles fuse only partially, releasing FcRn-IgG complexes to mix into the plasma membrane in cycles of 3-4s over prolonged periods of time.     

Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels

We have engineered the Fc region of a human immunoglobulin G (IgG) to generate a mutated antibody that modulates the concentrations of endogenous IgGs in vivo. This has been achieved by targeting the activity of the Fc receptor, FcRn, which serves through its IgG salvage function to maintain and regulate IgG concentrations in the body. We show that an IgG whose Fc region was engineered to bind with higher affinity and reduced pH dependence to FcRn potently inhibits FcRn-IgG interactions and induces a rapid decrease of IgG levels in mice. Such FcRn blockers (or 'Abdegs,' for antibodies that enhance IgG degradation) may have uses in reducing IgG levels in antibody-mediated diseases and in inducing the rapid clearance of IgG-toxin or IgG-drug complexes.     

IgG transcytosis and recycling by FcRn expressed in MDCK cells reveals ligand-induced redistribution

The neonatal Fc receptor (FcRn) transports IgG across epithelial cells and recycles serum IgG. FcRn binds IgG at the acidic pH of endosomes and releases IgG at the basic pH of blood. We expressed rat FcRn in polarized MDCK cells and demonstrated that it functions in transcytosis and recycling of IgG. In the absence of IgG, FcRn is distributed predominantly apically, but redistributes to basolateral locations upon IgG addition, indicating that ligand binding induces a signal that stimulates transcytosis. FcRn transcytoses IgG more efficiently in the apical to basolateral than the reverse direction when IgG is internalized by receptor-mediated endocytosis at acidic pH or by fluid phase endocytosis at basic pH. The PI 3-kinase inhibitor wortmannin disrupts basolateral recycling and transcytosis in both directions, but only minimally reduces apical recycling. Confocal imaging and quantitative IgG transport studies demonstrate that apically-internalized IgG recycles to the apical surface mainly from wortmannin-insensitive apical early endosomes, whereas FcRn-IgG complexes that transcytose to the basolateral surface pass through downstream Rab11-positive apical recycling endosomes and transferrin-positive common endosomal compartments.     

Generation of mutated variants of the human form of the MHC class I-related receptor,FcRn, with increased affinity for mouse immunoglobulin G

Much data support the concept that the MHC class I-related receptor FcRn serves to regulate immunoglobulin G (IgG) concentrations in serum and other diverse body sites in both rodents and humans. Previous studies have indicated that the human ortholog of FcRn is endowed with unexpectedly high stringency in binding specificity for IgGs. In contrast to mouse FcRn, which binds promiscuously to IgGs across species, human FcRn does not bind to mouse IgG1 or IgG2a, and interacts weakly with mouse IgG2b. Here, we investigate the molecular basis for this high-level specificity. We have systematically mutated human FcRn residues to the corresponding mouse FcRn residues in the regions that encompass the FcRn-IgG interaction site. Notably, mutation of the poorly conserved residue Leu137 of human FcRn to glutamic acid (L137E) generates a human FcRn mutant that binds to mouse IgG1 and mouse IgG2a with equilibrium dissociation constants of 13.2 microM and 14.4 microM, respectively. From earlier high-resolution structural analyses of the rat FcRn-rat Fc complex, residue 137 of human FcRn is predicted to contact residue 436 of IgG, which can be either His436 (mouse IgG1, mouse IgG2a) or Tyr436 (human IgG1, mouse IgG2b). The simplest interpretation of our data for the L137E mutant is therefore that replacement of the Leu137-Tyr436 (human) by the Glu137-His436 (mouse) pair generates a receptor that can bind to mouse IgG1 and mouse IgG2a. The L137E mutation reduces the affinity of human FcRn for human IgG1 by about twofold, consistent with the introduction of a less favorable Glu137-Tyr436 interaction. However, the analysis of the effects of other mutations on the binding to different IgGs indicates that the contribution to binding of the interaction of FcRn residue 137 with IgG residue 436 can vary. This suggests the existence of distinct docking topologies that are accompanied by variations in contacts between these two residues for different FcRn-IgG pairs. Our observations are of direct relevance to understanding the molecular nature of the human FcRn-IgG interaction. In turn, understanding human FcRn function has significance for the optimization of the serum half-lives of therapeutic and prophylactic antibodies.     

Neonatal FcR expression in bone marrow-derived cells functions to protect serum IgG from catabolism

The neonatal FcR (FcRn) is a receptor that protects IgG from catabolism and is important in maintaining high serum Ab levels. A major site of expression of FcRn is vascular endothelial cells where FcRn functions to extend the serum persistence of IgG by recycling internalized IgG back to the surface. Because FcRn is expressed in other tissues, it is unclear whether endothelial cells are the only site of IgG protection. In this study, we used FcRn-deficient mice and specific antiserum to determine the tissue distribution of FcRn in the adult mouse. In addition to its expression in the vascular endothelium of several organs, we found FcRn to be highly expressed in bone marrow-derived cells and professional APCs in different tissues. Experiments using bone marrow chimeras showed that FcRn expression in these cells acted to significantly extend the half-life of serum IgG indicating that in addition to the vascular endothelium, bone marrow-derived phagocytic cells are a major site of IgG homeostasis.     

Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium

The airway epithelium is a central player in the defense against pathogens including efficient mucociliary clearance and secretion of immunoglobulins, mainly polymeric IgA, but also IgG. Pulmonary administration of therapeutic antibodies on one hand, and intranasal immunization on the other, are powerful tools to treat airway infections. In either case, the airway epithelium is the primary site of antibody transfer. In various epithelia, bi-polar transcytosis of IgG and IgG immune complexes is mediated by the human neonatal Fc receptor, FcRn, but FcRn expression in the nasal epithelium had not been demonstrated, so far. We prepared affinity-purified antibodies against FcRn α-chain and confirmed their specificity by Western blotting and immunofluorescence microscopy. These antibodies were used to study the localization of FcRn α-chain in fixed nasal tissue. We here demonstrate for the first time that ciliated epithelial cells, basal cells, gland cells, and endothelial cells in the underlying connective tissue express the receptor. A predominant basolateral steady state distribution of the receptor was observed in ciliated epithelial as well as in gland cells. Co-localization of FcRn α-chain with IgG or with early sorting endosomes (EEA1-positive) but not with late endosomes/lysosomes (LAMP-2-positive) in ciliated cells was observed. This is indicative for the presence of the receptor in the recycling/transcytotic pathway but not in compartments involved in lysosomal degradation supporting the role of FcRn in IgG transcytosis in the nasal epithelium.     

Multivalent immune complexes divert FcRn to lysosomes by exclusion from recycling sorting tubules

The neonatal receptor for immunoglobulin G (IgG; FcRn) prevents IgG degradation by efficiently sorting IgG into recycling endosomes and away from lysosomes. When bound to IgG-opsonized antigen complexes, however, FcRn traffics cargo into lysosomes, where antigen processing can occur. Here we address the mechanism of sorting when FcRn is bound to multivalent IgG-opsonized antigens. We find that only the unbound receptor or FcRn bound to monomeric IgG is sorted into recycling tubules emerging from early endosomes. Cross-linked FcRn is never visualized in tubules containing the unbound receptor. Similar results are found for transferrin receptor, suggesting a general mechanism of action. Deletion or replacement of the FcRn cytoplasmic tail does not prevent diversion of trafficking to lysosomes upon cross-linking. Thus physical properties of the lumenal ligand–receptor complex appear to act as key determinants for sorting between the recycling and lysosomal pathways by regulating FcRn entry into recycling tubules.     

From sorting endosomes to exocytosis: association of Rab4 and Rab11 GTPases with the Fc receptor, FcRn, during recycling

A longstanding question in cell biology is how is the routing of intracellular organelles within cells regulated? Although data support the involvement of Rab4 and Rab11 GTPases in the recycling pathway, the function of Rab11 in particular is uncertain. Here we have analyzed the association of these two Rab GTPases with the Fc receptor, FcRn, during intracellular trafficking. This Fc receptor is both functionally and structurally distinct from the classical Fcgamma receptors and transports immunoglobulin G (IgG) within cells. FcRn is therefore a recycling receptor that sorts bound IgG from unbound IgG in sorting endosomes. In the current study we have used dual color total internal reflection fluorescence microscopy (TIRFM) and wide-field imaging of live cells to analyze the events in human endothelial cells that are involved in the trafficking of FcRn positive (FcRn(+)) recycling compartments from sorting endosomes to exocytic sites at the plasma membrane. Our data are consistent with the following model for this pathway: FcRn leaves sorting endosomes in Rab4(+)Rab11(+) or Rab11(+) compartments. For Rab4(+)Rab11(+) compartments, Rab4 depletion occurs by segregation of the two Rab proteins into discrete domains that can separate. The Rab11(+)FcRn(+) vesicle or tubule subsequently fuses with the plasma membrane in an exocytic event. In contrast to Rab11, Rab4 is not involved in exocytosis.     

Conferring the binding properties of the mouse MHC class I-related receptor, FcRn, onto the human ortholog by sequential rounds of site-directed mutagenesis

The MHC class I-related receptor, FcRn, is involved in binding and transporting immunoglobulin G (IgG) within and across cells. In contrast to mouse FcRn, which binds to IgGs from multiple different species, human FcRn is surprisingly stringent in binding specificity. For example, human FcRn does not bind to mouse IgG1 or IgG2a and interacts only weakly with mouse IgG2b. Here, we have used site-directed mutagenesis in combination with interaction (surface plasmon resonance) studies, with the goal of generating human FcRn variants that more closely resemble mouse FcRn in binding specificity. Our studies show that residues encompassing and extending away from the interaction site on the alpha2 helix of FcRn play a significant and most likely indirect role in FcRn-IgG interactions. Further, by combining mutations in the alpha2 helix with those in a non-conserved region of the alpha1 helix encompassing residues 79-89, we have generated a human FcRn variant that has properties very similar to those of mouse FcRn. These studies define the molecular basis for the marked difference in binding specificity between human and rodent FcRn, and give insight into how human FcRn recognizes IgGs.     

Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells

The neonatal Fc receptor, FcRn mediates an endocytic salvage pathway that prevents degradation of IgG, thus contributing to the homeostasis of circulating IgG. Based on the low affinity of IgG for FcRn at neutral pH, internalization of IgG by endothelial cells is generally believed to occur via fluid-phase endocytosis. To investigate the role of FcRn in IgG internalization, we used quantitative confocal microscopy to characterize internalization of fluorescent Fc molecules by HULEC-5A lung microvascular endothelia transfected with GFP fusion proteins of human or mouse FcRn. In these studies, cells transfected with FcRn accumulated significantly more intracellular Fc than untransfected cells. Internalization of FcRn-binding forms of Fc was proportional to FcRn expression level, was enriched relative to dextran internalization in proportion to FcRn expression level, and was blocked by incubation with excess unlabeled Fc. Because we were unable to detect either surface expression of FcRn or surface binding of Fc, these results suggest that FcRn-dependent internalization of Fc may occur through sequestration of Fc by FcRn in early endosomes. These studies indicate that FcRn-dependent internalization of IgG may be important not only in cells taking up IgG from an extracellular acidic space, but also in endothelial cells participating in homeostatic regulation of circulating IgG levels.     

Albumin binding to FcRn: distinct from the FcRn-IgG interaction

The MHC-related Fc receptor for IgG (FcRn) protects albumin and IgG from degradation by binding both proteins with high affinity at low pH in the acid endosome and diverting both from a lysosomal pathway, returning them to the extracellular compartment. Immunoblotting and surface plasmon resonance studies show that both IgG and albumin bind noncooperatively to distinct sites on FcRn, that the affinity of FcRn for albumin decreases approximately 200-fold from acidic to neutral pH, and that the FcRn-albumin interaction shows rapid association and dissociation kinetics. Isothermal titration calorimetry shows that albumin binds FcRn with a 1:1 stoichiometry and the interaction has hydrophobic features as evidenced by a large positive change in entropy upon binding. Our results suggest that the FcRn-albumin interaction has unique features distinct from FcRn-IgG binding despite the overall similarity in the pH-dependent binding mechanism by which both ligands are protected from degradation.     

Efficient mucosal vaccination mediated by the neonatal Fc receptor

Almost all infectious diseases are initiated at mucosal surfaces, yet intramuscular or subcutaneous vaccination usually provides only minimal protection at sites of infection owing to suboptimal activation of the mucosal immune system. The neonatal Fc receptor (FcRn) mediates the transport of IgG across polarized epithelial cells lining mucosal surfaces. We mimicked this process by fusing a model antigen, herpes simplex virus type-2 (HSV-2) glycoprotein gD, to an IgG Fc fragment. Intranasal immunization, together with the adjuvant CpG, completely protected wild-type, but not FcRn knockout, mice after intravaginal challenge with virulent HSV-2 186. This immunization strategy induced efficient mucosal and systemic antibody, B- and T-cell immune responses, with stable protection for at least 6 months after vaccination in most of the immunized animals. The FcRn-IgG transcellular transport pathway may provide a general delivery route for subunit vaccines against many mucosal pathogens.     

The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity

The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.     

Neonatal Fc receptor expression in macrophages is indispensable for IgG homeostasis

ABSTRACT

The maintenance of the homeostasis of immunoglobulin G (IgG) represents a fundamental aspect of humoral immunity that has direct relevance to the successful delivery of antibody-based therapeutics. The ubiquitously expressed neonatal Fc receptor (FcRn) salvages IgG from cellular degradation following pinocytic uptake into cells, conferring prolonged in vivo persistence on IgG. However, the cellular sites of FcRn function are poorly defined. Pinocytic uptake is a prerequisite for FcRn-mediated IgG salvage, prompting us to investigate the consequences of IgG uptake and catabolism by macrophages, which represent both abundant and highly pinocytic cells in the body. Site-specific deletion of FcRn to generate mice harboring FcRn-deficient macrophages results in IgG hypercatabolism and ~threefold reductions in serum IgG levels, whereas these effects were not observed in mice that lack functional FcRn in B cells and dendritic cells. Consistent with the degradative activity of FcRn-deficient macrophages, depletion of these cells in FcRn-deficient mice leads to increased persistence and serum levels of IgG. These studies demonstrate a pivotal role for FcRn-mediated salvage in compensating for the high pinocytic and degradative activities of macrophages to maintain IgG homeostasis.     

Stoichiometry of the interaction between the major histocompatibility complex-related Fc receptor and its Fc ligand.

The neonatal Fc receptor (FcRn) transports immunoglobulin G (IgG) across epithelia, providing passive immunity and protecting serum IgG from degradation. For both functions, FcRn binds to IgG at the acidic pH of intracellular vesicles (pH 相似文献   

Characterization of the rabbit neonatal Fc receptor (FcRn) and analyzing the immunophenotype of the transgenic rabbits that overexpresses FcRn

The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits--having one extra copy of the FcRn when hemizygous and two extra copies when homozygous--showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies.     

The MHC class II-associated invariant chain interacts with the neonatal Fc gamma receptor and modulates its trafficking to endosomal/lysosomal compartments

The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-beta(2)-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-gamma-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-gamma. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain.     

Ligand valency affects transcytosis, recycling and intracellular trafficking mediated by the neonatal Fc receptor

The neonatal Fc receptor (FcRn) transports IgG across epithelial cell barriers to provide maternal antibodies to offspring and serves as a protection receptor by rescuing endocytosed IgG and albumin from lysosomal degradation. Here we describe the generation of polarized Madin-Darby canine kidney (MDCK) cells expressing rat FcRn (rFcRn) to investigate the potential requirement for ligand bivalency in FcRn-mediated transport. The rFcRn-MDCK cells bind, internalize and bidirectionally transcytose the bivalent ligands IgG and Fc across polarized cell monolayers. However, they cannot be used to study FcRn-mediated transport of the monovalent ligand albumin, as we observe no specific binding, internalization or transcytosis of rat albumin. To address whether ligand bivalency is required for transport, the ability of rFcRn to transcytose and recycle wild-type Fc homodimers (wtFc; two FcRn-binding sites) and a heterodimeric Fc (hdFc; one FcRn-binding site) was compared. We show that ligand bivalency is not required for transcytosis or recycling, but that wtFc is transported more efficiently than hdFc, particularly at lower concentrations. We also demonstrate that hdFc and wtFc have different intracellular fates, with more hdFc than wtFc being trafficked to lysosomes and degraded, suggesting a role for avidity effects in FcRn-mediated IgG transport.     

Crystal structure and immunoglobulin G binding properties of the human major histocompatibility complex-related Fc receptor(,)

The neonatal Fc receptor (FcRn) performs two distinct but related functions: transport of maternal immunoglobulin G (IgG) to pre- or neonatal mammals, thus providing passive immunity, and protection of IgG from normal serum protein catabolism. FcRn is related to class I MHC proteins but lacks a functional peptide binding groove. The crystal structure of human FcRn has been determined at 2.7 A resolution and compared to the previously described structure of rat FcRn [Burmeister et al. (1994) Nature 372, 336-343] and to the structures of MHC and MHC-related proteins. Human FcRn is structurally similar to the rat receptor but does not form receptor dimers in the crystals as observed in crystals of rat FcRn. The interaction between human FcRn and IgG was characterized by determining the binding stoichiometry using equilibrium gel filtration and by deriving binding affinities for the different human IgG subclasses using a surface plasmon resonance assay. Like rat and mouse FcRn, human FcRn interacts with IgG with a 2:1 receptor:ligand stoichiometry. The binding of human FcRn to the four human IgG subclasses shows subclass and allotype variations but no clear subclass affinity differences that correlate with serum half-lives. The structure of human FcRn and studies of its ligand binding are relevant to current efforts to use FcRn-mediated regulation of IgG half-life in serum to increase the lifetimes of antibody-based therapeutics.