Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs

Maternal immunity is mediated exclusively by colostral immunoglobulins in ruminants. As the neonatal Fc receptor (FcRn) is suggested to be involved in the transport of immunoglobulin G (IgG) in the mammary gland, we cloned this receptor from sheep and analysed its expression in the mammary gland around the time of parturition and also in the small intestine from the newborn lamb. FcRn heavy-chain mRNA was detected (by using in situ hybridization) exclusively in the acinar and ductal epithelial cells in mammary gland biopsies both before and after parturition. Immunohistochemistry revealed that the cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition. A remarkable difference was observed in the pattern after lambing, where the apical side of the cells was strongly stained. The presence of the FcRn in the acinar and ductal epithelial cells of the mammary gland, and the obvious change in distribution before and after parturition, indicate that the FcRn plays an important role in the transport of IgG during colostrum formation in ruminants. Immunohistochemical analysis detected a strong apical and a weak basal FcRn signal in the duodenal crypt cells of a neonatal lamb, which have been previously demonstrated to secrete IgG1 in newborn ruminants. The FcRn was not detected in the duodenal enterocytes, which absorb intact IgG from the colostrum in a non-specific manner. These data suggest that FcRn is involved in IgG1 secretion in ruminant epithelial cells.     

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.     

Cloning and characterization of the dromedary (Camelus dromedarius) neonatal Fc receptor (drFcRn)

The full length cDNA of the dromedary neonatal Fc receptor (drFcRn) chain was isolated and found that it is similar to the neonatal Fc receptor (FcRn) of other species with a high overall similarity to ruminant FcRn chains. The drFcRn/Fc contact residues are highly conserved and predicted to bind both conventional (IgG1) and heavy chain (IgG2a, IgG3) antibodies. Using immunohistochemistry, we detected its expression in the hepatocytes and in epithelial cells of portal bile ductuli and also in the mammary gland acini and ducti. Remarkably, Ser313, that was identified to be crucial for apical to basolateral transcytosis, is substituted in the drFcRn chain. The full length of the dog and orangutan FcRn chains was also identified from databases. Analyzing the phylogenetic relatedness of this gene we found that dromedary clustered together with artiodactyls, dog is located between artiodactyls and primates, where the orangutan was branched, reflecting the accepted evolutionary relationships.     

Expression of the Fc receptor in the mammary gland during lactation in the marsupial Trichosurus vulpecula (brushtail possum)

One of several functions described for the Fc receptor is regulation of IgG isotype transport into milk. The first marsupial homologues of the Fc receptor heavy and light chains, FcRn and beta-2 microglobulin, from the brushtail possum have been cloned and characterised. The level of FcRn mRNA in the possum mammary gland was highest at the start of lactation, and decreased slowly thereafter. Expression of FcRn mRNA did not increase during the switch phase when the concentration of IgG in milk is highest. In contrast, the level of beta-2 microglobulin mRNA in the mammary gland increased during the switch phase when milk IgG concentration also increases. This correlation between beta-2 microglobulin mRNA expression in the mammary gland with the time of active IgG-transfer into milk was also observed in the bovine and murine mammary gland. This suggests that expression of the Fc receptor in the mammary gland is controlled by the expression of beta-2 microglobulin and that its expression is upregulated during the period of highest IgG-transfer into milk.     

Nonclassical major histocompatibility complex I-like Fc neonatal receptor (FcRn) expression in neonatal human tissues

The neonatal Fc receptor (FcRn) was demonstrated to play a role both in the recycling and thus the protection of immunoglobulin G (IgG) from catabolism and in the maternal-fetal transfer of IgG. The expression of this particular receptor was evidenced in a variety of cell types, but the endothelial cell was considered the main cell able to perform both recycling and IgG catabolism. Based on preliminary data obtained in adult human mammary glands and skin, this study focused on a number of neonatal human tissues, targeting FcRn expression mainly in epithelial versus endothelial cells. Our results demonstrate that in most of the investigated tissues, the neonatal Fc receptor is not detectable in the endothelial cells lining the capillaries, whereas most epithelial cells are positive. We could also observe the receptor's expression in most macrophages, smooth muscle cells, and neurons. Taken together, these data suggest that the main sites of IgG catabolism might in fact be other than endothelial cells in human neonates.     

IgG transport across mucosal barriers by neonatal Fc receptor for IgG and mucosal immunity

Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain significant quantities of IgG. The neonatal Fc receptor for IgG (FcRn) plays a major role in regulating host IgG levels and transporting IgG and associated antigens across polarized epithelial barriers. The FcRn can then recycle the IgG/antigen complex back across the intestinal barrier into the lamina propria for processing by dendritic cells and presentation to CD4⁺ T cells in regional organized lymphoid structures. FcRn, through its ability to secrete and absorb IgG, thus integrates luminal antigen encounters with systemic immune compartments and, as such, provides essential host defense and immunoregulatory functions at the mucosal surfaces.     

Neonatal Fc Receptor: From Immunity to Therapeutics

The neonatal Fc receptor (FcRn), also known as the Brambell receptor and encoded by Fcgrt, is a MHC class I like molecule that functions to protect IgG and albumin from catabolism, mediates transport of IgG across epithelial cells, and is involved in antigen presentation by professional antigen presenting cells. Its function is evident in early life in the transport of IgG from mother to fetus and neonate for passive immunity and later in the development of adaptive immunity and other functions throughout life. The unique ability of this receptor to prolong the half-life of IgG and albumin has guided engineering of novel therapeutics. Here, we aim to summarize the basic understanding of FcRn biology, its functions in various organs, and the therapeutic design of antibody- and albumin-based therapeutics in light of their interactions with FcRn.     

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.     

The MHC class I-related receptor, FcRn, plays an essential role in the maternofetal transfer of gamma-globulin in humans

The transfer of maternal gamma-globulin (IgG) provides the neonate with humoral immunity during early life. In humans, maternal IgG is transported across the placenta during the third trimester of pregnancy. The expression of the MHC class I-related receptor, FcRn, in the human placenta suggests that this Fc receptor might be involved in the delivery of maternal IgG, but direct evidence to support this is lacking. In the current study an ex vivo placental model has been used to analyze the maternofetal transfer of a recombinant, humanized (IgG1) antibody in which His435 has been mutated to alanine (H435A). In vitro binding studies using surface plasmon resonance indicate that the mutation ablates binding of the antibody to recombinant mouse and human FcRn. Relative to the wild-type antibody, the H435A mutant is deficient in transfer across the placenta. Significantly, the mutation does not affect binding to Fc gamma RIII, an FcR that has been suggested in earlier studies to mediate the transfer of maternal IgG. The analyses demonstrate that binding of an IgG to FcRn is a prerequisite for transport across the perfused placenta. FcRn therefore plays a central role in the maternofetal delivery of IgG and this has implications for the use of protein engineering to improve the properties of therapeutic antibodies.     

Neonatal Fc receptors discriminates and monitors the pathway of native and modified immunoglobulin G in placental endothelial cells

In the placenta, immunoglobulin G (IgG) is selectively transported from mother to fetus by a highly regulated transcellular mechanism aimed to achieve fetal humoral immunity. We questioned the role of neonatal Fc receptors (FcRn) in the traffic of IgG in human placental endothelial cells (HPEC). Cells were cultured in a double-chamber system and further exposed to IgG or Fc or to diethylpyrocarbonate-modified IgG or Fc in which the receptor recognition domain of the molecule (CH2-CH3) was altered. We provide evidence that the native IgG/Fc probes are transcytosed or recycled by HPEC, whereas the probes with the altered receptor recognition domain (which do not bind to FcRn) massively accumulate into the endocytic/lysosomal compartments. The results indicate that FcRn distinguishes between the intact and modified IgG and control their cellular traffic: native IgG is salvaged and released out of the cells, whereas modified IgG is retained and sorted to a degradative pathway. The data advance the understanding of the basic mechanism for IgG traffic in human endothelial cells, which may be exploited for the specific transport of antibodies in various immune disorders.     

A strategy for bacterial production of a soluble functional human neonatal Fc receptor

The major histocompatibility complex (MHC) class I related receptor, the neonatal Fc receptor (FcRn), rescues immunoglobulin G (IgG) and albumin from lysosomal degradation by recycling in endothelial cells. FcRn also contributes to passive immunity by mediating transport of IgG from mother to fetus (human) or newborn (rodents), and may translocate IgG over mucosal surfaces. FcRn interacts with the Fc-region of IgG and domain III of albumin with binding at pH 6.0 and release at pH 7.4. Knowledge of these interactions has facilitated design of recombinant proteins with altered serum half-lives and/or altered biodistribution. To generate further research in this field, there is a great need for large amounts of soluble human FcRn (shFcRn) for in vitro interaction studies. In this report, we describe a novel laboratory scale production of functional shFcRn in Escherichia coli (E. coli) at milligram level. Truncated wild type hFcRn heavy chains were expressed, extracted, purified from inclusion bodies under denaturing non-reducing conditions, and subsequently refolded in the presence of human beta(2)-microglobulin (hbeta(2)m). The secondary structural elements of refolded heterodimeric shFcRn were correctly formed as demonstrated by circular dichroism (CD). Furthermore, functional and stringent pH dependent binding to IgG and human serum albumin were demonstrated by ELISA and surface plasmon resonance (SPR). This method may be easily adapted for the expression of large amounts of other FcRn species and MHC class I related molecules.     

Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn.

The analysis of the pharmacokinetics of wild-type and mutated Fc fragments derived from human IgG1 indicates that Ile253, His310 and His435 play a central role in regulating serum half-life in mice. Reduced serum half-life of the recombinant, mutated fragments correlates with decreased binding to the MHC class I-related neonatal Fc receptor, FcRn. In addition, the analysis of an Fc fragment in which His435 is mutated to Arg435 demonstrates that the sequence difference at this position between human IgG1 (His435) and IgG3 (Arg435) most likely accounts for the shorter serum half-life of IgG3 relative to IgG1. In contrast to His310 and His435, the data indicate that His433 does not play a role in regulating the serum half-life of human IgG1. Thus, the interaction site of mouse FcRn on human and mouse IgG1 involves the same conserved amino acids located at the CH2-CH3 domain interface of the IgG molecule. The sequence similarities between mouse and human FcRn suggest that these studies have direct relevance to understanding the factors that govern the pharmacokinetics of therapeutic IgG.     

FcRn is not the receptor mediating the transfer of serum IgG to colostrum in pigs

In contrast to humans or rabbits, in which maternal IgG is transmitted to offspring prenatally via the placenta or the yolk sac, large domestic animals such as pigs, cows and sheep transmit IgG exclusively through colostrum feeding after delivery. The extremely high IgG content in colostrum is absorbed by newborns via the small intestine. Although it is widely accepted that the neonatal Fc receptor, FcRn, is the receptor mediating IgG transfer across both the placenta and small intestine, it remains unclear whether FcRn also mediates serum IgG transfer across the mammary barrier to colostrum/milk, especially in large domestic animals. In this study, using a FcRn knockout pig model generated with a CRISPR‐Cas9‐based approach, we clearly demonstrate that FcRn is not responsible for the IgG transfer from serum to colostrum in pigs, although like in other mammals, it is involved in IgG homeostasis and mediates IgG absorption in the small intestine of newborns.     

In situ phosphorylation of Akt and ERK1/2 in rat mammary gland, colon, and liver following treatment with human insulin and IGF-1

High doses of insulin and the insulin analog AspB10 have been reported to increase mammary tumor incidence in female rats likely via receptor-mediated mechanisms, possibly involving enhanced IGF-1 receptor activation. However, insulin and IGF-1 receptor functionality and intracellular signaling in the rat mammary gland in vivo is essentially unexplored. The authors investigated the effect of a single subcutaneous dose of 600 nmol/kg human insulin or IGF-1 on Akt and ERK1/2 phosphorylation in rat liver, colon, and mammary gland. Rat tissues were examined by Western blotting and immunohistochemistry by phosphorylation-specific antibodies. Insulin as well as IGF-1 caused Akt phosphorylation in mammary epithelial cells, with myoepithelial and basal epithelial cells being most sensitive. IGF-1 caused stronger Akt phosphorylation than insulin in mammary gland epithelial cells. Phosphorylation of ERK1/2 was not influenced by insulin or IGF-1. Rather, in liver and mammary gland P-ERK1/2 appeared to correlate with estrous cycling, supporting that ERK1/2 has important physiological roles in these two organs. In short, these findings supported that the rat mammary gland epithelium expresses functional insulin and IGF-1 receptors and that phosphorylation of Akt as well as ERK1/2 may be of value in understanding the effects of exogenous insulin in the rat mammary gland and colon.     

Lymphocyte homing and Ig secretion in the murine mammary gland

In mice the majority of the immunoglobulins (Ig) in milk belongs to the IgA class. Prior to its transepithelial transportation into the milk, dimeric IgA (dIgA) is bound to the transmembrane form of the secretory component or polymeric Ig receptor (SC/pIgR). The latter is synthesized in the epithelial cells lining the ducts and alveoli of the mammary gland. A candidate for playing the role of adhesion molecule to primed lymphocytes present in the murine mammary gland might be the mucosal addressin cell adhesion molecule-1 (MAdCAM-1). We studied the correlation between the levels of IgA in colostrum and milk, the number of IgA producing plasma cells in the mammary gland and the expression of MAdCAM-1 in mammary gland endothelial cells during pregnancy and lactation. The relation between the IgA levels in the milk and the expression levels of pIgR in mammary gland epithelial cells was also investigated. We found that the expression of MAdCAM-1 and pIgR starts in early-mid pregnancy; the number of IgA-producing plasma cells and the IgA concentration in milk increase from early lactation onwards. The MAdCAM-1 expression declines during lactation whereas the pIgR levels and IgA-producing plasma cell numbers rise until the end of lactation. Because the MAdCAM-1 level starts to rise several days before the rise of the IgA-producing plasma cell level, MAdCAM-1 cannot be the rate determining factor governing extravasation of primed B cells to the mammary gland. We also conclude that the pIgR is present in sufficient amounts to enable increasing S-IgA secretion into the milk during lactation.     

Malignant myoepithelioma with a squamous epithelial component in the mammary gland of a cynomolgus monkey

A solid mass arising from the mammary gland was found in a 7-year-old female cynomolgus monkey. Histologically, the mass consisted of 2 components: spindle-shaped or ovoid sarcomatous cells and squamous epithelial cells. Metastatic nodules noted in the lung, liver and the gallbladder had the same histological features as the mammary mass. Immunohistochemistry revealed that the sarcomatous cells were positive for alpha-smooth muscle actin (alpha-SMA), vimentin, calponin, S-100 protein, epithelial membranous antigen (EMA), cytokeratin (large spectrum) and cytokeratin 14 (CK 14) in the cytoplasm, and p53, erbB-2 and progesterone receptor in the nuclei, but negative for desmin and estrogen receptor. The squamous epithelial cells were positive for EMA, cytokeratin (large spectrum) and CK 14, but negative for the rest. Both sarcomatous and squamous epithelial components were negative for glial fibrillary acidic protein (GFAP). Based on histological and immunohistochemical features, the present case was diagnosed as a malignant myoepithelioma with a squamous epithelial component in the mammary gland with distant metastases.     

Expression of functionally active FcRn and the differentiated bidirectional transport of IgG in human placental endothelial cells

The mechanism of selective transport of the immunoglobulins G from the placental stroma to the lumen of the fetal blood vessels has not been elucidated yet. It was postulated that the specific transport as well as the regulation of IgG level in the blood, involves the MHC class I related receptor FcRn for the Fc domain of IgG. We questioned whether human placental endothelial cells (HPEC) express FcRn and, if present, whether it is in a functionally active form. The experiments were performed on cultured HPEC and as positive control, human trophoblastic (JEG3) and mouse endothelial cells (SVEC) were used. Expression of FcRn, was demonstrated by indirect immunofluorescence and RT-PCR. The role of FcRn was assessed by quantifying the transcellular transport of [(125)I]-hIgG or [(125)I]-rF(ab')(2) fragments from the apical to basolateral surface, and in the reverse direction of HPEC grown on filters in a double chamber system. The intracellular pathway of FcRn or IgG was examined by electron microscopy using the proteins adsorbed to 5 nm and 20 nm colloidal gold particles, respectively. The results showed that: (a) FcRn is expressed by human placental endothelial cells, in a functionally active form; (b) transcytosis of IgG in HPEC is a time-dependent process that takes place preferentially from the basolateral to the apical compartment; and (c) both IgG and FcRn colocalize in an intracellular endocytic compartment, chloroquine sensitive. Together these data suggest that the regulation of IgG level by endothelial cells may result from interplay between salvaging, exocytosis, and transcytosis of the molecules. One can assume that IgG that does not bind to FcRn may be destined for destruction, and this would explain the mechanism by which IgG homeostasis is maintained.     

Characterization of surface receptors on bovine leukocytes

Three bovine leukocyte populations--peripheral blood lymphocytes (PBL), mammary gland polymorphonuclear neutrophils (PMN) and macrophages (Mo)--were characterized with respect to five surface markers: surface immunoglobulin (SIg), sheep erythrocyte receptor, complement (C) receptor and Fc receptors for both IgG and IgM. The majority of PMN and Mo possessed C and Fc receptors for IgG, but lacked SIg and the erythrocyte receptor. The PMN, but not Mo, also expressed a Fc receptor for IgM. The PBL were heterogeneous with respect to their surface characteristics and evidence was presented for the following subtypes: (a) cells with the E receptor alone; (b) cells with E receptor plus the Fc(IgG) receptor; (c) cells with SIg plus the C receptor but minus the Fc(IgG) receptor; (d) lymphocytes with SIg plus the C receptor and the Fc(IgG) receptor, and (e) cells lacking E receptors and SIg but bearing Fc(IgG). It was assumed, but not proven, that some of these latter cells must also bear the C receptor. The significance of the various cell types in antiviral defense is briefly discussed.