FcRn Alpha-chain Research Articles

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.

Position independent and copy-number-related expression of the bovine neonatal Fc receptor α-chain in transgenic mice carrying a 102 kb BAC genomic fragment

We generated and characterized transgenic mice carrying a 102 kb bovine genomic fragment, encoding the neonatal Fc receptor alpha-chain (bFcRn). FcRn plays a crucial role in the maternal IgG transport and it also regulates the IgG and albumin homeostasis. Some of its functions and transcriptional regulation show species specific differences. The FcRn heterodimer is composed of the alpha-chain and beta-2-microglobulin (beta2 m). A bacterial artificial chromosome containing the bovine FcRn alpha-chain gene (bFCGRT) with its 44 kb 5' and 50 kb long 3' flanking sequences was microinjected into fertilized mouse oocytes. Two of the transgenic lines generated, showed copy number related and integration site independent bFcRn expression. The bFcRn alpha-chain forms a functional receptor with the mouse beta2-microglobulin and extends the half-life of the mouse IgG in transgenic mice. Our results underline the feasibility of creating BAC transgenic mouse models of economically important bovine genes.

Isolation of the gene encoding the bovine neonatal Fc receptor

The role of an IgG-Fc receptor (FcRn) that resembles a class I MHC Ag in transporting IgGs through epithelial cells was recently shown in selected species. Here we report our preliminary characterization of a clone encoding the alpha chain of the bovine FcRn from a BAC library. The recombinant BAC DNA was digested, analyzed by Southern blot hybridization and a bovine FcRn positive 9 kb long fragment was subcloned and partially sequenced. The exon/intron organization of the bovine FcRn alpha chain gene was deduced by comparison with its cDNA sequence. The sequence revealed a similar organization to the human and mouse FcRn alpha chain genes. The bovine FcRn alpha chain gene has acquired several repetitive sequences in its 5′-flanking region, including multiple SINE and LINE elements. Potential binding sites for transcription factors within the 5′-flanking sequence were identified using TESS and TFSEARCH programs. The 5′-flanking region of the bFcRn alpha chain gene was analyzed for its ability to directly express the luciferase reporter gene in bovine mammary gland epithelial cells. Transient transfection of a luciferase construct revealed that there was promoter activity in the region −1787 to +92 of the 5′-flanking sequence.

Expression of FcRn, the MHC Class I-Related Receptor for IgG, in Human Keratinocytes

The neonatal Fc receptor (FcRn) for IgG has been shown to be responsible for IgG transport and to be involved in IgG catabolism. In this study, we show expression of FcRn in normal human epidermal keratinocytes. By RT-PCR, we demonstrate the FcRn alpha-chain mRNA obtained from cultured keratinocytes creating a 457 bp product as confirmed by sequence analysis. Northern blot analysis shows a 1.5 kb transcript. Real-time PCR reveals consistent expression of FcRn alpha-chain mRNA in human keratinocytes from different donors. Anti-FcRn alpha2-extracellular domain and anti-FcRn cytoplasmic tail antibody (Ab) directed against defined antigenic targets were generated and used for immunoblotting and immunoprecipitation revealing protein expression of the 46 kDa FcRn alpha-chain. By immunofluorescence microscopy, we find granular-vesicular staining for FcRn alpha-chain in keratinocytes. Fluorescence-activated cell sorting analysis gives predominantly an intracellular distribution of FcRn in keratinocytes. Biochemically, we demonstrate Fc-dependent binding of human IgG at acidic pH. In normal human epidermis, we find a cytoplasmic vesicular staining of predominantly basal and suprabasal keratinocytes. In summary, we demonstrate expression of a functional FcRn in normal human epidermal keratinocytes. These findings further emphasize the role of keratinocytes as immunomodulating cells in inflammatory and immunologic processes of the skin.

Localization of the sheep FcRn in the mammary gland

Among the multiple functions, which have been identified for the neonatal Fc receptor (FcRn), we study its role in the IgG transport in the mammary gland during the colostrum formation. For this reason, we have obtained several mammary gland biopsies from a pregnant sheep around parturition. The presence of the FcRn heavy chain mRNA was detected exclusively in the acinar and ductal epithelial cell by in situ hybridization (ISH). We detected strong signal in samples harvested 24 and 10 days prepartum; however, in samples we collected postpartum was barely detectable. Immunohistochemistry confirmed our ISH data. The cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition, although a remarkable difference was observed in the pattern after lambing. The signal indicated uneven distribution of the FcRn alpha chain in the epithelial cells 1 and 5 days postpartum, since the apical sides of the epithelial cells were highlighted. The presence of the FcRn in the acinar and ductal epithelial cells and the obvious change of its distribution before and after parturition suggest that FcRn plays an important role in the IgG transport during colostrum formation. FcRn expression was also found in the lamb duodenal crypt epithelial cells, which have been previously demonstrated to secrete IgG1 in newborn ruminants, suggesting secretory role of the FcRn in ruminant epithelial cells.

Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice.

The MHC class I-related receptor, FcRn, mediates the transfer of maternal gamma globulin (IgG) to young rodents, primarily via intestinal transcytosis, and this provides humoral immunity for the first few weeks after birth. In a previous study, the site of mouse IgG1 (mIgG1) with which FcRn interacts has been mapped using recombinant wild-type and mutated Fc-hinge fragments. The site encompasses residues at the CH2-CH3 domain interface of Fc (Ile253, His310, Gln311, His433 and Asn434) and the same amino acids are involved in regulating the pharmacokinetics of the Fc-hinge fragments. This suggests that in addition to its known function, FcRn might also play a role in IgG homeostasis. Consistent with this hypothesis, in this study, we demonstrate that FcRn alpha-chain mRNA is present not only in neonatal brush border but also in other tissues of adult animals (liver, lung, spleen and endothelial cells). In addition, analysis of the pharmacokinetics of mouse Ig/Fc-hinge fragments in genetically manipulated mice that are deficient in the expression of FcRn demonstrates that the beta-phase half-lives are abnormally short. These findings suggest that FcRn is involved in IgG homeostasis.

Requirement for a beta 2-microglobulin-associated Fc receptor for acquisition of maternal IgG by fetal and neonatal mice.

There is considerable evidence to suggest that an FcR similar in structure to class I MHC Ags, neonatal Fc receptor (FcRn), transports IgG across the intestinal epithelium of suckling mice. However, this has not previously been shown definitively, nor has it been shown whether FcRn is the only, or even the major, IgG transporter in the neonatal mouse gut. We report here that neonatal mice homozygous for a targeted disruption of the beta 2microglobulin (beta 2m) gene, which encodes one subunit of FcRn, had reduced FcRn alpha-chain at the lumenal plasma membrane of intestinal cells. These mice had strikingly lower serum IgG levels during the first month after birth than littermates that possessed functional FcRn. Furthermore, we found by fostering mice on mothers with a different IgG allotype that all of the IgG in sera of beta 2m-/- mice was endogenous, and that none was obtained from milk. We conclude that FcRn is the only transporter of IgG from mother to young in the mouse. The onset of IgG synthesis in mice that received no milk IgG lagged behind that in siblings with normal IgG transport, suggesting that maternal IgG stimulates Ab production in the neonate. We noted no difference between the IgG concentrations in the milk of beta 2m-/- and beta 2m+/- mice, indicating that FcRn is not involved in the secretion of IgG into milk.

Mouse MHC class I-like Fc receptor encoded outside the MHC.

In many mammalian species antibodies transmitted from the mother provide humoral immunity to the young. Maternal IgG from milk is transported across the intestinal epithelium of neonatal rats by an Fc receptor (FcRn) that comprises an alpha-chain similar to the class I Ag of the MHC and beta 2-microglobulin. Suckling mice also acquire antibodies by uptake from the gut. We made a neonatal mouse intestinal cDNA library and screened it with a probe encoding rat FcRn alpha-chain. The nucleotide and predicted amino acid sequences of the two positive clones were very similar to those of rat FcRn. Comparison of the FcRn domains to various MHC class I and CD1 molecules suggests a divergence of FcRn from MHC early in the mammalian lineage. We expressed one of these cDNA in mouse 3T3 fibroblasts. Cells that expressed the cDNA product bound the Fc fragment of IgG with the same pH dependence as neonatal rat intestinal epithelium. We detected RNA that hybridize with the mouse cDNA only in neonatal small intestine and fetal yolk sac, two tissues involved in IgG transport. These data show that the mouse cDNA code for FcRn alpha-chain. The mouse FcRn alpha-chain is similar in sequence to the class I MHC Ag, encoded on chromosome 17 in the mouse. However, we find that the mouse FcRn gene lies outside the MHC, on chromosome 7.