The Bidirectional Transfer and Fetal Vascular Pressure Changes Due to the Presence of [Formula: see text] I-Labeled Inhibin A in the ex-vivo Human Placental Model

Objective: The purpose of this study was to investigate the transport of inhibin A and to determine its effects on fetal vascular pressure at elevated levels in the human placenta using (125)I -labeled synthetic glycoprotein. Methods: Synthetic inhibinAwas prepared and was shown to be consistent with the natural form by high-pressure liquid chromatography (HPLC) and molecular weight determination by gas-chromatography mass spectrometry. The standardized Na(125)I process yielded (125)I -labeled inhibin A with a radioactivity of 10(6) cpm/μg. This compound was placed in the human placenta in maternal–fetal and fetal–maternal studies using antipyrine and (14)C -labeled inulin as controls to determine the bidirectional transfer of the compound. Results: Maternal–fetal and fetal–maternal clearance indices were 0.045± 0.003 and 0, respectively. In eight placentas there was no evidence of vascular pressure changes due to the presence of up to 5000 pg of inhibin A. Conclusions: There is minimal maternal–fetal transfer and no detectable fetal–maternal transfer in normotensive and pregnancy-induced hypertensive placentas. In addition, there are no pressure changes in the fetal vascular system due to the clinically significant levels of inhibin A

Transcytosis and catabolism of antibody

This review describes the evolution of our knowledge of the transmission of immunoglobulin G (IgG) from mother to infant and the factors which regulate the persistence of IgG in the circulation. These apparently unrelated processes involve the same Fc receptor, FcRn (n = neonatal). FcRn appears to carry out these diverse roles by binding to IgG and then either transporting the bound IgG across cells (transcytosis) or recycling its cargo back to the cell surface (control of catabolism). IgG that is taken up by cells in the absence of binding to FcRn undergoes degradation. Thus, FcRn is the "protective" receptor that serves to maintain IgG homeostasis and deliver IgGs across cellular barriers.</p

FcRn: The MHC class I-related receptor that is more than an IgG transporter

Recent data have indicated that the major histocompatibility complex (MHC) class I-related receptor FcRn plays a role in regulating serum IgG levels, in addition to transferring maternal IgGs across the rodent neonatal gut. The isolation of a human homolog of FcRn from placenta suggests that the studies in rodents have relevance to understanding similar processes in humans. This has implications for the engineering of improved antibodies for therapy.</p

Multiple roles for the major histocompatibility complex class I-related receptor FcRn

Multiple functions have recently been identified for the neonatal Fc receptor FcRn. In addition, a human homolog of the rodent forms of FcRn has been identified and characterized. This major histocompatibility complex class I-related receptor plays a role in the passive delivery of immunoglobulin (Ig)Gs from mother to young and the regulation of serum IgG levels. In addition, FcRn expression in tissues such as liver, mammary gland, and adult intestine suggests that it may modulate IgG transport at these sites. These diverse functions are apparently brought about by the ability of FcRn to bind IgGs and transport them within and across cells. However, the molecular details as to how FcRn traffics within cells have yet to be fully understood, although in vitro systems have been developed for this purpose. The molecular nature of the FcRn-IgG interaction has been studied extensively and encompasses residues located at the CH2-CH3 domain interface of the Fc region of IgG. These Fc amino acids are highly conserved in rodents and man and interact with residues primarily located on the α2 domain of FcRn. Thus, it is now possible to engineer IgGs with altered affinities for FcRn, and this has relevance to the modulation of IgG serum half-life and maternofetal IgG transport for therapeutic applications.</p

Evidence to support the cellular mechanism involved in serum IgG homeostasis in humans

IgG is the most abundant serum antibody and is an essential component of the humoral immune response. It is known that the 'neonatal' Fc receptor (FcRn) plays a role in maintaining constant serum IgG levels by acting as a protective receptor which binds and salvages IgG from degradation. However, the cellular mechanism that is involved in serum IgG homeostasis is poorly understood. In the current study we address this issue by analyzing the intracellular fate in human endothelial cells of IgG molecules which bind with different affinities to FcRn. The studies show that IgG which do not bind to FcRn accumulate in the lysosomal pathway, providing a cellular explanation for short serum persistence of such antibodies. We have also investigated the saturability of the homeostatic system and find that it has limited capacity. Our observations have direct relevance to the understanding and treatment of IgG deficiency, and to the effective application of therapeutic antibodies.</p

Delineation of the Amino Acid Residues Involved in Transcytosis and Catabolism of Mouse IgG1

The MHC class I-related receptor, FcRn, is involved in both the transcytosis of serum γ-globulins (IgGs) and in regulating their serum persistence. The interaction site of FcRn on the Fc region of rodent IgG has been mapped to residues at the CH2-CH3 domain interface using site-directed mutagenesis and x-ray crystallographic analyses. In the current study, the role of individual residues (H310, H433, and N434) at this interface in mediating the Fc-FcRn interaction has been investigated using recombinant, mutated Fc hinge fragments derived from mouse IgG1. In addition, two highly conserved Fc histidines (H435 and H436) have been mutated to alanine, and the resulting mutated Fc hinge fragments were analyzed in both transcytosis and pharmacokinetic studies in mice and in competition binding assays using recombinant, soluble FcRn. The analyses indicate that mutation of H310, H435, and, to a lesser extent, H436 to alanine results in reduced activity of the Fc hinge fragments in both in vivo and in vitro assays. Thus, in addition to the previously defined role of I253 in the FcRn-IgG interaction, these histidines play a key role in mediating the functions conducted by this Fc receptor. The effects of these mutations on binding of Fc hinge fragments to staphylococcal protein A have also been analyzed and demonstrate a partial, but not complete, overlap of the FcRn and staphylococcal protein A interaction sites on mouse IgG1.</p

Evidence that the hinge region plays a role in maintaining serum levels of the murine IgG1 molecule

The site of the murine IgG1 molecule that regulates catabolism has recently been shown to encompass amino acids that are located at the CH2CH3 domain interface. The CH2 and CH3 domains are connected to each other by a relatively flexible "mini-hinge" region, and flexibility in this region could clearly affect the orientation of the domains with respect to each other. The internal movement of the CH2 domain depends on the absence/presence of the hinge disulphide. The increased mobility of the CH2 domain relative to the CH3 domain in a hingeless IgG or Fc fragment may result in a conformational change at the CH2CH3 domain interface and alter the accessibility of the residues that are involved in catabolism control. To investigate this possibility, four Fc fragments which differ in the presence/absence of hinge disulphides and hinge sequences have been analysed in both in vivo pharmacokinetic studies and in vitro by limited proteolysis with pepsin. The data show that the presence of hinge disulphide(s) in the Fc fragment results in a longer intravascular half life but a higher susceptibility to pepsin attack. This, taken together with the knowledge that pepsin cleaves close to the CH2CH3 domain interface, suggests that the longer half life of disulphide linked Fc fragments relative to unlinked fragments may be due to conformational differences in this region of the IgG molecule, and these conformational changes may affect the accessibility of the catabolic site for binding to putative protective Fc receptors.</p

Differences in promiscuity for antibody-FcRn interactions across species: Implications for therapeutic antibodies

Preclinical tests of therapeutic antibodies are frequently carried out in mice to evaluate pharmacokinetics and efficacy. However, the observation that mouse IgG are cleared rapidly from the human circulation suggests that mice may not always be an ideal model. The Fc receptor, FcRn, regulates the serum half-lives of IgG in mice and most likely has a similar function in humans. In the current study we have carried out an extensive analysis of the interaction of the human or mouse forms of FcRn with IgG from various species using surface plasmon resonance. We show that in contrast to mouse FcRn, human FcRn is surprisingly stringent in its binding specificity for IgG derived from different species. Human FcRn binds to human, rabbit and guinea pig IgG, but not significantly to rat, bovine, sheep or mouse IgG (with the exception of weak binding to mouse IgG2b). In contrast, mouse FcRn binds to all IgG analyzed. The lack of binding of human FcRn to mouse IgG1 has been confirmed using transfectants that have been engineered to express human FcRn on the cell surface. Our results provide a molecular explanation for the enigmatic observation that mouse IgG behave anomalously in humans. These studies have implications for the successful application of therapeutic antibodies.</p

Identification and function of neonatal Fc receptor in mammary gland of lactating mice

In addition to its proposed function in regulating serum IgG levels, the MHC class I-related neonatal Fc receptor (FcRn) is known to play a role in IgG transfer across rodent yolk sac and neonatal intestine. In contrast to humans, for which transplacental transfer of IgG appears to be the only mechanism of maternal IgG delivery, the transmission of IgG in mice occurs both antenatally (yolk sac) and neonatally (transport from mother's milk across intestinal epithelial cells). In the current study, a possible role for FcRn in regulating IgG transfer into milk has been investigated. FcRn has been shown to be present in functional form in the mammary gland of lactating mice, and is localized to the epithelial cells of the acini. Analysis of the transfer of Fc fragments and IgG which have different affinities for FcRn indicate that, unexpectedly, these proteins are transferred in inverse correlation with their binding affinity for FcRn. Thus, in the lactating mammary gland FcRn appears to play a role in recycling IgG in a mode that may have relevance to FcRn trafficking during the maintenance of constant serum IgG levels.</p