Immunoglobulin G Transport Research Articles

Immunomodulation of Antibody Glycosylation through the Placental Transfer

Establishing an immune balance between the mother and fetus during gestation is crucial, with the placenta acting as the epicenter of immune tolerance. The placental transfer of antibodies, mainly immunoglobulin G (IgG), is critical in protecting the developing fetus from infections. This review looks at how immunomodulation of antibody glycosylation occurs during placental transfer and how it affects fetal health. The passage of maternal IgG antibodies through the placental layers, including the syncytiotrophoblast, stroma, and fetal endothelium, is discussed. The effect of IgG subclass, glycosylation, concentration, maternal infections, and antigen specificity on antibody transfer efficiency is investigated. FcRn-mediated IgG transport, influenced by pH-dependent binding, is essential for placental transfer. Additionally, this review delves into the impact of glycosylation patterns on antibody functionality, considering both protective and pathological effects. Factors affecting the transfer of protective antibodies, such as maternal vaccination, are discussed along with reducing harmful antibodies. This in-depth examination of placental antibody transfer and glycosylation provides insights into improving neonatal immunity and mitigating the effects of maternal autoimmune and alloimmune conditions.

4 Identifying the immunoglobulin G transporter in equine tissues: A look at the neonatal Fc receptor

The importance of immunoglobulin G (IgG) is well established regarding its contribution to maintaining health status. However, the mechanism for controlling IgG movement through tissues is less known. The neonatal Fc receptor (FcRn) has been identified as the receptor responsible for bidirectional transport of IgG across cells, maintenance of IgG levels in serum, and assisting with antigen presentation. Unfortunately, little is known about the FcRn in livestock. Therefore, the objective of this study was to provide critical fundamental information regarding the location of the FcRn in equine tissues, focusing on those with key health importance. It was our hypothesis that FcRn abundance would be highest in respiratory and gastrointestinal (GI) tissues. Tissues were collected from 6 horses of mixed breed, age (3–22yr), and sex (male n = 4; female n = 2) immediately following euthanasia for various chronic health conditions at North Dakota State University (n = 2) or Texas A&M University (n = 4). Sampling locations and procedures were standardized across universities to minimize variation. Tissue locations included the respiratory tract, GI tract, other visceral organs, the cornea, and the synovial membrane of the stifle and carpal joints. Tissues collected were fixed in 10% NBF before embedding in paraffin and cross sectioning at 5μm. Tissue cross sections were mounted on glass slides, stained for the FcRn, and counterstained with DAPI. Areas of interest were captured using the tiling module of Zeiss ZEN software and the Plan-Apochromat 20x/0.8 NA lens. Images were analyzed using Image Pro software with data represented as relative fluorescence (RF) within areas of interest to represent FcRn abundance. Data were analyzed using the general linear model procedure of SAS.Relative fluorescence differed between tissue types (P < 0.01). The highest RF was seen in joint synovium and the respiratory tract. Moderate RF was seen in GI tissues and the lowest RF was seen in other visceral organs. These data lay the foundation for all future studies regarding the FcRn and IgG and their role in disease prevention and treatment.

In silicooptimization of prenatal vaccine regimens to promote early-life immunity

Abstract Newborn children are vulnerable to infection due to their dampened humoral immune response. Maternal immunoglobulin G (IgG) transferred across the placenta provides passive immunity to newborns, and prenatal vaccination to boost pathogen-specific IgG transfer successfully reduced the incidence of neonatal tetanus, diphtheria, and acellular pertussis (Tdap) infections. Despite this, higher rates of infection among premature neonates signifies a deficiency in current immunization programs. To identify regulators of IgG transfer and refine prenatal vaccine regimens, we used a novel in silico approach. To disentangle the role of placental Fc gamma receptors (FcγRs) in IgG transfer, we conducted multiplexed immunofluorescence (mIF) to measure FcγR expression in paraffin-embedded placental tissue. A mechanistic model of placental IgG transfer informed by single cell RNA sequencing and the mIF data revealed FcγRIIb as a key driver of dynamic subclass-specific IgG transcytosis in placental endothelial cells (ECs). In model simulations and experiments in vitro with human umbilical vein ECs, we found the maternal IgG profile fine-tunes antigen- and subclass-specific IgG transfer dynamics. To identify immunization windows of opportunity for maximum IgG transfer, we simulated maternal vaccination by integrating a model of B cell activation and antibody production into the mechanistic model. This revealed first trimester vaccination as a potential strategy to maximize Tdap-induced IgG transport to premature newborns. We conclude that optimal prenatal vaccine regimens depend on gestational length and that novel prenatal vaccines can leverage these determinants of IgG transport to maximize neonatal immune protection. Supported by grants from NIH (T32 GM145443) the Jeffress Trust Awards Program.

PSII-A-6 Evaluation of Maternal Plane of Nutrition and Selenium Supplementation on the Neonatal Fragment Crystallizable Receptor (Fcrn) in ewe Mammary Tissue

Abstract Maternal immunoglobulin G (IgG) in livestock species is transferred to the offspring exclusively through colostrum. In ruminants, a highly selective mammary barrier allows only immunoglobulin G1 to enter colostrum in substantial amounts. The neonatal fragment crystallizable receptor (FcRn) is responsible for the bidirectional transport of IgG; however, its role in concentrating IgG into colostrum is not well described. The objective of this study was to evaluate maternal nutrition and supranutritional selenium (Se) supplementation on FcRn abundance in mammary tissue. Pregnant Rambouillet ewes (n = 80) were used in a 2 x 3 factorial treatment arrangement which included the main effects of dietary Se [adequate Se (9.5 µg/kg of BW) vs. high Se (81.8 µg/kg of BW)] and plane of nutrition [60%, 100%, and 140% of requirements]. After parturition, ewe mammary glands were collected, and tissue samples were fixed in 10% NBF before paraffin embedding and sectioning. Cross-sections (5 μm) were mounted onto glass slides, deparaffinized, stained for FcRn, and counterstained with DAPI. Areas of tissue cross-sections were captured with Plan-Apochromat 20x/0.8 NA lens using the tiling module of Zeiss software. Images were analyzed using ImagePro Premiere software. Relative fluorescence within the areas of interest was used as an indicator of FcRn abundance with data presented as receptor abundance per glandular tissue area. Data were analyzed using the general linear model procedure of SAS. High Se supplementation increased FcRn abundance per glandular area (P = 0.01). Abundance of mammary FcRn was not influenced by maternal plane of nutrition. Mammary FcRn was positively correlated (r2 = 0.25; P = 0.02) with IgG concentration in colostrum. Results indicate the presence of FcRn in ewe mammary tissue and correlation with colostral IgG. Further evaluation of the role of supranutritional Se and the FcRn is needed.

Research progress on neonatal Fc receptor and its application.

Neonatal Fc receptor (FcRn) is a specific receptor for immunoglobulin G (IgG) and albumin, which binds to them in a pH-dependent manner and prevents them from lysosomal degradation to keep a long plasma half-life. In addition, FcRn plays an important role in transmembrane transport of IgG and albumin and in antigen presentation. In autoimmune diseases, anti-FcRn antibody can promote the degradation of pathogenic IgG by competitive binding to FcRn. In infectious diseases, the half-life of drugs can be prolonged by increasing the affinity between therapeutic antibody and FcRn, while the combination of viral antigen and Fc fragment of IgG can cause local immune response of mucosa for disease prevention and treatment. In cancer, albumin as a carrier of anticancer drugs can achieve efficient drug delivery, and FcRn itself may be used as a predictor of the prognosis of cancer patients. This review details the functions of FcRn, highlights its role in autoimmune diseases, infectious diseases and cancer, as well as the mechanism of drug development based on FcRn, to provide a reference for the clinical application and drug development of FcRn.

An IgA mimicry of IgG that binds Polymeric Immunoglobulin Receptor for mucosa transcytosis.

Most pathogens establish infection through mucosa, where secretory immunoglobulin A (sIgA) plays an ‘immune exclusion’ role in humoral defense. Extravasation of intravenously (i.v.) administrated therapeutic immunoglobulin G (IgG) mainly relies on convection and/or neonatal Fc receptor-mediated transcytosis from circulation into interstitial space. Active transport of interstitial IgG further across epithelium into mucosa, like sIgA, is a much desired feature for the next generation of therapeutic antibodies, especially for anti-infection purposes. For the first time, we report the engineering of an IgA mimicry of IgG, with its Fc portion in fusion with the 18-aa tail piece (tp) of sIgA and the J chain, possessing sIgA’s full binding activity towards polymeric immunoglobulin receptor that mediates mucosa transcytosis. In a diphtheria toxin receptor (DTR) knockin mouse model, i.v. injected anti-diphtheria toxin (DT) IgG(tp)J protected DTR+ cells from deletion upon DT injection. The compact design of IgG(tp)J opens new revenues for more effective therapeutic IgG mimicking some of the important biological functions of IgA.

Intravenous immunoglobulin significantly reduces exposure of concomitantly administered anti-C5 monoclonal antibody tesidolumab.

Awareness of drug-drug interactions is critical in organ transplant recipient management. However, biologic agents interfering with monoclonal antibodies is not widely considered. We report the effect of high-dose intravenous immunoglobulin (IVIg) on safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of the human anti-C5 monoclonal antibody tesidolumab (LFG316) in end-stage renal disease patients awaiting kidney transplant. In this single-center, phase 1, open-label, parallel-group study, 8 patients were assigned to receive either single-dose tesidolumab+IVIg or tesidolumab alone, with 56-day follow-up. Within-group PK parameters were consistent. Mean tesidolumab exposure decreased 34%, clearance increased 63%, and half-life decreased 41% comparing tesidolumab+IVIg to tesidolumab alone. IVIg influence on tesidolumab elimination was most evident in the first 3weeks. Complete suppression of both total and alternative complement activities was maintained for 4weeks in the tesidolumab alone group and for 2weeks in the tesidolumab+IVIg group. Tesidolumab was well tolerated. IVIg infused before tesidolumab affected tesidolumab PK and PD, resulting in a shortened period of full complement activity inhibition. These findings suggest a clinically relevant impact of IVIg on monoclonal antibody clearance and indirectly hint at an IVIg mechanism of action in treating autoimmune diseases and allosensitization by accelerating pathogenic IgG antibody degradation. Trial registration number: NCT02878616.

ImmunoglobulinG transport increases in an in vitro blood-brain barrier model with amyloid-β and with neuroinflammatory cytokines.

Immunotherapies are a promising strategy for the treatment of neurological diseases such as Alzheimer's disease (AD), however, transport of antibodies to the brain is severely restricted by the blood-brain barrier (BBB). Furthermore, molecular transport at the BBB is altered in disease, which may affect the mechanism and quantity of therapeutic antibody transport. To better understand the transport of immunotherapies at the BBB in disease, an in vitro BBB model derived from human induced pluripotent stem cells (iPSCs) was used to investigate the endocytic uptake route of immunoglobulinG (IgG). In this model, uptake of fluorescently labeled IgGs is a saturable process. Inhibition of clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis demonstrated that macropinocytosis is a major transport route for IgGs at the BBB. IgG uptake and transport were increased after the addition of stimuli to mimic AD (Aβ1-40 and Aβ1-42 ) and neuroinflammation (tumor necrosis factor-α and interleukin-6). Lastly, caveolar endocytosis increased in the AD model, which may be responsible for the increase in IgG uptake in disease. This study presents an iPSC-derived BBB model that responds to disease stimuli with physiologically relevant changes to molecular transport and can be used to understand fundamental questions about transport mechanisms of immunotherapies in health and neurodegenerative disease.

Noncanonical placental Fc receptors: What is their role in modulating transplacental transfer of maternal IgG?

The transplacental transfer of maternal Immunoglobulin G (IgG) to the fetus is critical for protection against infectious diseases in the first year of life [1]. Maternal protective IgG is transferred from the maternal to the fetal circulatory system via the placenta, and this process begins in the first trimester of pregnancy [2]. By 37–40 weeks of gestation, maternal passively acquired IgG concentrations in newborns can exceed maternal IgG serum levels in normal pregnancies [3–7]. Yet, the molecular mechanisms of transplacental transfer of maternal IgG remain poorly understood. In order to reach the fetal circulatory system, maternal IgG must traverse three distinct placental anatomical barriers: (1) the syncytiotrophoblast cell barrier, (2) the villous stroma containing placental fibroblasts and Hofbauer cells, and (3) fetal endothelial cells. It is well established that IgG crosses the syncytiotrophoblast by binding to the canonical IgG shuttle receptor: Fragment crystallizable (Fc) receptor neonatal (FcRn) [2, 8]. However, how maternal IgG traverses the subsequent placental barriers is not completely understood, as they do not express FcRn, yet recent RNAseq analyses have shown that Fcγ receptors, including FcγRIIIa, FcγRIIa, FcγRIIb, and FcγRI, are expressed in term placentas [9]. However, it should be cautioned that it is not yet known if these noncanonical placental FcRs play a role, if at all, in the transplacental transfer of maternal IgG. A deeper understanding of the molecular mechanism(s) of IgG binding to placentally expressed Fc receptors could be important (1) for the design of novel maternal IgG-based therapeutics and vaccines with optimal transplacental transfer efficiency, with the ultimate goal of increasing infant protection against congenital and neonatal infectious diseases, and (2) to optimize the Fc region of immunomodulatory IgG monoclonal antibody therapeutics for blunted transplacental transfer to potentially reduce the transplacental transport of maternal self-reactive IgG in women with autoimmune disorders.

Delivery of immunoglobulin G antibodies to the rat nervous system following intranasal administration: Distribution, dose-response, and mechanisms of delivery

The intranasal route has been hypothesized to circumvent the blood-brain and blood-cerebrospinal fluid barriers, allowing entry into the brain via extracellular pathways along olfactory and trigeminal nerves and the perivascular spaces (PVS) of cerebral blood vessels. We investigated the potential of the intranasal route to non-invasively deliver antibodies to the brain 30 min following administration by characterizing distribution, dose-response, and mechanisms of antibody transport to and within the brain after administering non-targeted radiolabeled or fluorescently-labeled full length immunoglobulin G (IgG) to normal adult female rats. Intranasal [125I]-IgG consistently yielded highest concentrations in the olfactory bulbs, trigeminal nerves, and leptomeningeal blood vessels with their associated PVS. Intranasal delivery also resulted in significantly higher [125I]-IgG concentrations in the CNS than systemic (intra-arterial) delivery for doses producing similar endpoint blood concentrations. Importantly, CNS targeting significantly increased with increasing dose only with intranasal administration, yielding brain concentrations that ranged from the low-to-mid picomolar range with tracer dosing (50 μg) up to the low nanomolar range at higher doses (1 mg and 2.5 mg). Finally, intranasal pre-treatment with a previously identified nasal permeation enhancer, matrix metalloproteinase-9, significantly improved intranasal [125I]-IgG delivery to multiple brain regions and further allowed us to elucidate IgG transport pathways extending from the nasal epithelia into the brain using fluorescence microscopy. The results show that it may be feasible to achieve therapeutic levels of IgG in the CNS, particularly at higher intranasal doses, and clarify the likely cranial nerve and perivascular distribution pathways taken by antibodies to reach the brain from the nasal mucosae.

Allogenic Fc Domain-Facilitated Uptake of IgG in Nasal Lamina Propria: Friend or Foe for Intranasal CNS Delivery?

Background: The use of therapeutic antibodies for the treatment of neurological diseases is of increasing interest. Nose-to-brain drug delivery is one strategy to bypass the blood brain barrier. The neonatal Fc receptor (FcRn) plays an important role in transepithelial transcytosis of immunoglobulin G (IgG). Recently, the presence of the FcRn was observed in nasal respiratory mucosa. The aim of the present study was to determine the presence of functional FcRn in olfactory mucosa and to evaluate its role in drug delivery. Methods: Immunoreactivity and messenger RNA (mRNA) expression of FcRn was determined in ex vivo porcine olfactory mucosa. Uptake of IgG was performed in a side-by-side cell and analysed by immunofluorescence. Results: FcRn was found in epithelial and basal cells of the olfactory epithelium as well as in glands, cavernous bodies and blood vessels. Allogenic porcine IgGs were found time-dependently in the lamina propria and along axonal bundles, while only small amounts of xenogenic human IgGs were detected. Interestingly, lymphoid follicles were spared from allogenic IgGs. Conclusion: Fc-mediated transport of IgG across the nasal epithelial barrier may have significant potential for intranasal delivery, but the relevance of immune interaction in lymphoid follicles must be clarified to avoid immunogenicity.

Intranasal delivery of antibodies bypasses the blood‐brain barrier and results in significantly higher central nervous system levels than systemic administration

Passive immunotherapy has become a prominent therapeutic strategy for the treatment of many different neurological disorders. Antibodies can be targeted to the pathogenic proteins of diseases such as Alzheimer's (e.g., beta‐amyloid and phosphorylated tau), Parkinson's (e.g., alpha‐synuclein), and multiple sclerosis (e.g., CD20 marker on B cells). However, delivery of antibodies to the central nervous system (CNS) following systemic administration is significantly impeded as a result of the blood‐brain barrier (BBB) and blood‐cerebrospinal‐fluid barriers (BCSFBs). The intranasal route has been hypothesized to circumvent the BBB and BCSFBs and allow entry into the brain via extracellular pathways along cranial nerves and the perivascular spaces (PVS) of cerebral blood vessels (Thorne et al. Neuroscience 2004 &amp; 2008; Lochhead et al. J. Cereb. Blood Flow &amp; Metab. 2015). Here, we demonstrate the potential of intranasal delivery as a non‐invasive strategy to bypass the BBB and BCSFBs to rapidly target full length immunoglobulin G (IgG) antibodies to the CNS. Quantitative distribution studies of radiolabeled IgG in the CNS of anesthetized rats revealed that intranasal delivery achieved significantly higher antibody levels in both the CNS and CSF compared to systemic delivery at doses resulting in matched blood exposure via either route. Additional experiments exploring the intactness of radiolabeled IgG in the brain following intranasal delivery were conducted to identify the biochemical conditions necessary for IgG intactness in vivo. Finally, by utilizing fluorophore‐labeled IgG, we provide direct evidence of intranasal IgG transport along perineural and perivascular pathways that directly connect the nasal mucosa to CNS target sites. Our results suggest olfactory and trigeminal nerve‐associated perineural channels provide direct pathways to the olfactory bulbs and brainstem, respectively. Once within the brain, further distribution along perivascular spaces of cerebral blood vessels is capable of transporting IgG to widespread regions of the brain. Our findings highlight that the intranasal route can provide a non‐invasive, direct, and rapid method for targeting antibodies to the CNS, bypassing the BBB and BCSFBs in a way that merits further consideration for the treatment of neurological diseases.Support or Funding InformationSupported by the University of Wisconsin‐Madison School of Pharmacy, the Graduate School at the University of Wisconsin, the Michael J. Fox Foundation for Parkinson's Research, the Parkinson's Foundation, the Wisconsin Alzheimer's Disease Research Center (NIH P50‐AG033514), the Wisconsin institute for Clinical and Translational Research (Clinical and Translational Science Award program through the NIH National Center for Advancing Translational Sciences, grant UL1TR000427), and the Wisconsin Alumni Research Foundation grant 135 PRJ82AZ.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The expression and function of the neonatal Fc receptor in thyrocytes of Hashimoto's thyroiditis

ObjectiveThyroglobulin (Tg) and thyroid peroxidase (TPO) antibodies (TgAb and TPOAb), which are primarily of the immunoglobulin G (IgG) class, can mediate antibody-dependent cell-mediated cytotoxicity in vitro. However, it is unclear whether any thyrocyte molecules can facilitate the transport and elimination of TgAb and TPOAb. The IgG transport receptor neonatal Fc receptor (FcRn) is a candidate mediator of these processes. In this study, we aimed to evaluate FcRn expression and function in normal and Hashimoto's thyroiditis (HT) thyrocytes. MethodsFcRn expression in primary thyrocyte cultures (four normal and four HT groups) was examined by polymerase chain reaction (PCR) and Western blotting. Localization of FcRn was demonstrated by immunoelectron microscopy. A double immunofluorescence staining method was adopted to detect FcRn and internalized human TgAb IgG. Stimulation experiments were performed to assess the regulation of FcRn expression by T helper cell 1 (Th1) (IFN-γ and TNF-α) and Th2 cytokines (IL-10 and IL-4). ResultsFcRn expression was lower in HT thyrocytes than in normal thyrocytes. FcRn was located in the cytoplasm, membranes, mitochondria and transport vesicles of thyrocytes. Both human IgG and TgAb IgG were internalized by thyrocytes in a pH-dependent manner and co-localized with FcRn in thyrocytes. FcRn expression was downregulated by Th1 and Th2 cytokines in both normal and HT thyrocytes in a dose-dependent manner. ConclusionsOur results suggest that FcRn may be associated with the transport and metabolism of IgG in thyrocytes and that transport is independent of IgG type. FcRn may be involved in HT pathogenesis.

Probing the extracellular diffusion of antibodies in brain using in vivo integrative optical imaging and ex vivo fluorescence imaging

Antibody-based therapeutics exhibit great promise in the treatment of central nervous system (CNS) disorders given their unique customizable properties. Although several clinical trials have evaluated therapeutic antibodies for treatment of CNS disorders, success to date has likely been limited in part due to complex issues associated with antibody delivery to the brain and antibody distribution within the CNS compartment. Major obstacles to effective CNS delivery of full length immunoglobulin G (IgG) antibodies include transport across the blood–brain and blood–cerebrospinal fluid barriers. IgG diffusion within brain extracellular space (ECS) may also play a role in limiting central antibody distribution; however, IgG transport in brain ECS has not yet been explored using established in vivo methods. Here, we used real-time integrative optical imaging to measure the diffusion properties of fluorescently labeled, non-targeted IgG after pressure injection in both free solution and in adult rat neocortex in vivo, revealing IgG diffusion in free medium is ~10-fold greater than in brain ECS. The pronounced hindered diffusion of IgG in brain ECS is likely due to a number of general factors associated with the brain microenvironment (e.g. ECS volume fraction and geometry/width) but also molecule-specific factors such as IgG size, shape, charge and specific binding interactions with ECS components. Co-injection of labeled IgG with an excess of unlabeled Fc fragment yielded a small yet significant increase in the IgG effective diffusion coefficient in brain, suggesting that binding between the IgG Fc domain and endogenous Fc-specific receptors may contribute to the hindered mobility of IgG in brain ECS. Importantly, local IgG diffusion coefficients from integrative optical imaging were similar to those obtained from ex vivo fluorescence imaging of transport gradients across the pial brain surface following controlled intracisternal infusions in anesthetized animals. Taken together, our results confirm the importance of diffusive transport in the generation of whole brain distribution profiles after infusion into the cerebrospinal fluid, although convective transport in the perivascular spaces of cerebral blood vessels was also evident. Our quantitative in vivo diffusion measurements may allow for more accurate prediction of IgG brain distribution after intrathecal or intracerebroventricular infusion into the cerebrospinal fluid across different species, facilitating the evaluation of both new and existing strategies for CNS immunotherapy.

IgG subclasses and allotypes: from structure to effector functions.

Of the five immunoglobulin isotypes, immunoglobulin G (IgG) is most abundant in human serum. The four subclasses, IgG1, IgG2, IgG3, and IgG4, which are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. These regions are involved in binding to both IgG-Fc receptors (FcγR) and C1q. As a result, the different subclasses have different effector functions, both in terms of triggering FcγR-expressing cells, resulting in phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement. The Fc-regions also contain a binding epitope for the neonatal Fc receptor (FcRn), responsible for the extended half-life, placental transport, and bidirectional transport of IgG to mucosal surfaces. However, FcRn is also expressed in myeloid cells, where it participates in both phagocytosis and antigen presentation together with classical FcγR and complement. How these properties, IgG-polymorphisms and post-translational modification of the antibodies in the form of glycosylation, affect IgG-function will be the focus of the current review.

Expression of Neonatal Fc Receptor in the Eye

The neonatal Fc receptor (FcRn) plays a critical role in the homeostasis and degradation of immunoglobulin G (IgG). It mediates the transport of IgG across epithelial cell barriers and recycles IgG in endothelial cells back into the bloodstream. These functions critically depend on the binding of FcRn to the Fc domain of IgG. The half-life and distribution of intravitreally injected anti-VEGF molecules containing IgG-Fc domains might therefore be affected by FcRn expressed in the eye. In order to establish whether FcRn-Fc(IgG) interactions may occur in the eye, we studied the mRNA and protein distribution of FcRn in postmortem ocular tissue. We used qPCR to study mRNA expression of the transmembrane chain of FcRn (FCGRT) in retina, optic nerve, RPE/choroid plexus, ciliary body/iris plexus, lens, cornea, and conjunctiva isolated from mouse, rat, pig, and human postmortem eyes and used immunohistochemistry to determine the pattern of FcRn expression in FCGRT-transgenic mouse and human eyes. In all four tested species, Fcgrt mRNA was expressed in the retina, RPE/choroid, and the ciliary body/iris, while immunohistochemistry documented FcRn protein expression in the ciliary body epithelium, macrophages, and endothelial cells in the retinal and choroidal vasculature. Our results demonstrate that FcRn has the potential to interact with IgG-Fc domains in the ciliary epithelium and retinal and choroidal vasculature, which might affect the half-life and distribution of intravitreally injected Fc-carrying molecules.

Neonatal Fc Receptor and its Role in the Absorption, Distribution, Metabolism and Excretion of Immunoglobulin G-Based Biotherapeutics

The neonatal Fc receptor (FcRn) is a heterodimeric membrane associated protein expressed in a variety of endothelial, epithelial and hematopoietic cells. FcRn regulates pH dependent intracellular trafficking of immunoglobulin G (IgG) and albumin, resulting in enhanced serum persistence and transcellular permeability of these proteins compared to other proteins of similar size. FcRn confers passive immunity during the early stages of life by facilitating maternal transmission of antibodies during gestation, and in some species during the neonatal period. The receptor continues to contribute to immunity beyond the perinatal period and throughout life by providing immunosurveillance in intestinal, pulmonary and genitourinary mucosa. In this capacity, FcRn facilitates bidirectional transport of IgG across mucosa and intracellular trafficking of antigen-antibody complexes in antigen presenting cells. Based on the functional roles of FcRn in regulating serum persistence and transcellular permeability, protein engineers have sought to exploit this receptor as a means of enhancing the absorption, distribution, metabolism and excretion (ADME) of IgG-based therapeutics. In this review, the current state of knowledge regarding the structural, mechanistic and functional properties of FcRn, as they relate to the ADME of IgG-based therapeutics, are discussed.

Contribution of FcRn binding to intestinal uptake of IgG in suckling rat pups and human FcRn-transgenic mice

Immunoglobulin G (IgG) is transcytosed across intestinal epithelial cells of suckling mammals by the neonatal Fc receptor (FcRn); however, the contribution of FcRn vs. FcRn-independent uptake to serum IgG levels had not been determined in either rat pups or human (h)FcRn-expressing mice (Tg276 and Tg32). In isoflurane-anesthetized rodents, serum levels were determined after regional intestinal delivery of human monoclonal antibodies (hIgG) with either wild-type (WT) Fc sequences or variants engineered for different FcRn binding affinities. Detection of full-length hIgG was by immunoassay; intestinal hFcRn and hIgG localization was by immunocytochemistry. High (μg/ml) serum levels of hIgG were detected after proximal intestinal delivery (0.1-10 mg/kg) in 2-wk-old rats. Human FcRn was visualized in epithelial cells of Tg276 mice, but low serum hIgG levels (<10 ng/ml) were obtained. In rat pups, intraintestinal hIgG1 WT administration resulted in dose-related and saturable uptake, whereas uptake of a low FcRn-binding affinity variant was nonsaturable. There were no differences in hIgG levels from systemic and hepatic portal vein serum samples, and intense hIgG immunostaining was noted in villi enterocytes and within lymphatic lacteal-like vessels. This study demonstrated that FcRn-mediated uptake in rat pups accounted for ~80% of serum hIgG levels and that IgG enters the circulation via the lymph and not the hepatic portal vein. The remaining uptake though the immature intestine is nonreceptor mediated. Intestinal epithelial cell hFcRn expression occurred in Tg276 mice, but receptor-mediated transport of IgG was not observed. The suckling rat pup intestine is a mechanistic model of FcRn-IgG-mediated transcytosis.

FcRn mediated IgG transport by retinal pigment epithelium cells

Background The neonatal Fc receptor (FcRn) binds immunoglobulin G (IgG), protects it from degradation and can transport IgG from apical to basolateral side of epithelial cells [1]. Previously, we showed that human retinal pigment epithelial (RPE) cells express FcRn and that, in contrast to non-ocular cells, TNF-a downregulates FcRn expression in RPE cells [2]. We speculated that FcRn in RPE plays a pivotal role in ocular IgG metabolism. The aim of this study was to develop a method to investigate whether RPE cells transport IgG molecules.

Tissues Expression, Polymorphisms Identification of FcRn Gene and Its Relationship with Serum Classical Swine Fever Virus Antibody Level in Pigs.

Neonatal Fc receptor (FcRn) gene encodes a receptor that binds the Fc region of monomeric immunoglobulin G (IgG) and is responsible for IgG transport and stabilization. In this report, the 8,900 bp porcine FcRn genomic DNA structure was identified and putative FcRn protein included 356 amino acids. Alignment and phylogenetic analysis of the porcine FcRn amino acid sequences with their homologies of other species showed high identity. Tissues expression of FcRn mRNA was detected by real time quantitative polymerase chain reaction (Q-PCR), the results revealed FcRn expressed widely in ten analyzed tissues. One single nucleotide polymorphism (SNP) (HQ026019:g.8526 C>T) in exon6 region of porcine FcRn gene was demonstrated by DNA sequencing analysis. A further analysis of SNP genotypes associated with serum Classical Swine Fever Virus antibody (anti-CSFV) concentration was performed in three pig populations including Large White, Landrace and Songliao Black pig (a Chinese indigenous breed). Our results of statistical analysis showed that the SNP had a highly significant association with the level of anti-CSFV antibody (At d 20; At d 35) in serum (p = 0.008; p = 0.0001). Investigation of expression and polymorphisms of the porcine FcRn gene will help us in further understanding the molecular basis of the antibody regulation pathway in the porcine immune response. All these results indicate that FcRn gene might be regarded as a molecular marker for genetic selection of anti-CSFV antibody level in pig disease resistance breeding programmes.