Mice homozygous for a β2-microglobulin gene disruption do not express any detectable β2-m protein. They express little if any functional major histocompatibility complex (MHC) class I antigen on the cell surface yet are fertile and apparently healthy. They show a normal distribution of γδ, CD4 + 8 + and CD4 + 8 - T cells, but have no mature CD4 - 8 + T cells and are defective in CD4 - 8 + T cell-mediated cytotoxicity. Our results strongly support earlier evidence that MHC class I molecules are crucial for positive selection of T cell antigen receptor αβ + CD4 - 8 + T cells in the thymus and call into question the non-immune functions that have been ascribed to MHC class I molecules.

Mice homozygous for a beta 2-microglobulin gene disruption do not express any detectable beta 2-m protein. They express little if any functional major histocompatibility complex (MHC) class I antigen on the cell surface yet are fertile and apparently healthy. They show a normal distribution of gamma delta, CD4(+)8(+) and CD4(+)8(-) T cells, but have no mature CD4(-)8(+) T cells and are defective in CD4(-)8(+) T cell-mediated cytotoxicity. Our results strongly support earlier evidence that MHC class I molecules are crucial for positive selection of T cell antigen receptor alpha beta(+) CD4(-)8(+). T cells in the thymus and call into question the non-immune functions that have been ascribed to MHC class I molecules.

The neonatal Fc receptor for immunoglobulin G (IgG) (FcRn) functions to transport maternal IgG to the fetal/neonatal animals and protects IgG from catabolism. The present study identified two pFcRn cDNAs (1.071 and 0.795 kb) from intestinal epithelial cells. The corresponding mRNA transcripts were detected in porcine kidney cell line LLC-PK1, peripheral blood mononuclear cells, and porcine tissues by reverse transcription-PCR (RT-PCR) and Northern blot. Sequence analysis showed that the 1.071 kb cDNA encodes the full-length pFcRn (pFcRn-L), whereas the 0.795 kb cDNA codes for a truncated pFcRn (pFcRn-S) with deletion of 92 amino acids matching the α2 domain of pFcRn-L. pFcRn-L was constitutively expressed by epithelial cells; however, pFcRn-S was not detectable in porcine tissues and cell lines although its transcript was abundant. Despite the lack of native pFcRn-S, pFcRn-S was readily detected in transfected cells. Recombinant pFcRn-L was confirmed to bind IgG at pH 6.0, but not at pH 7.5; however, pFcRn-S failed to bind IgG at both pH 5.0–6.0 and 7.5. pFcRn-L was expressed on the cell surface and mainly localized in early endosomes. In contrast, pFcRn-S was absent from cell surface and primarily localized in the lysosome and pFcRn-S trafficking to lysosomes was independent of β2-microglobulin (β2m). The accumulation of pFcRn-S in the lysosome may explain the absence of native pFcRn-S protein expression. In addition, the trafficking of pFcRn-S to the lysosomal compartment suggests that in addition to sorting signals in its cytoplasmic tail, the FcRn structural integrity may be important for proper intracellular trafficking and function.

Expression of many MHC genes is enhanced at the transcriptional or posttranscriptional level following exposure to the cytokine IFN-gamma. However, in this study we found that IFN-gamma down-regulated the constitutive expression of the neonatal Fc receptor (FcRn), an MHC class I-related molecule that functions to transport maternal IgG and protect IgG and albumin from degradation. Epithelial cell, macrophage-like THP-1 cell, and freshly isolated human PBMC exposure to IFN-gamma resulted in a significant decrease of FcRn expression as assessed by real-time RT-PCR and Western blotting. The down-regulation of FcRn was not caused by apoptosis or the instability of FcRn mRNA. Chromatin immunoprecipitation and gel mobility shift assays showed that STAT-1 bound to an IFN-gamma activation site in the human FcRn promoter region. Luciferase expression from an FcRn promoter-luciferase reporter gene construct was not altered in JAK1- and STAT-1-deficient cells following exposure to IFN-gamma, whereas expression of JAK1 or STAT-1 protein restored the IFN-gamma inhibitory effect on luciferase activity. The repressive effect of IFN-gamma on the FcRn promoter was selectively reversed or blocked by mutations of the core nucleotides in the IFN-gamma activation site sequence and by overexpression of the STAT-1 inhibitor PIAS1 or the dominant negative phospho-STAT-1 mutations at Tyr-701 and/or Ser-727 residues. Furthermore, STAT-1 might down-regulate FcRn transcription through sequestering the transcriptional coactivator CREB binding protein/p300. Functionally, IFN-gamma stimulation dampened bidirectional transport of IgG across a polarized Calu-3 lung epithelial monolayer. Taken together, our results indicate that the JAK/STAT-1 signaling pathway was necessary and sufficient to mediate the down-regulation of FcRn gene expression by IFN-gamma.

Orrie M. Friedmana, Paul Matsudairab, Arthur H. Reis, Jr.c,d, Neil Simisterc,e and Thomas Pochapskyd,e,∗ GrenPharma LLC, Waltham, MA, USA Whitehead Institute, Massachusetts Institute of Technology, Cambridge, MA, USA Department of Biology, Brandeis University, Waltham, MA, USA Department of Chemistry MS 015, Brandeis University, MA, USA Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USA

The neonatal Fc receptor, FcRn, transports proteins through cells, avoiding degradative compartments. FcRn is used in many physiological processes where proteins must remain intact while they move through cells. These contexts include the transport of IgG antibodies from mother to offspring, and the protection of IgG and albumin from catabolism. In polarized cell models, FcRn in the plasma membrane is predominantly at the basolateral surface. This distribution depends on two signals that overlap endocytosis signals. One of these signals resembles a YXXΦ motif, but with a tryptophan in place of the critical tyrosine residue; the other is a DDXXXLL signal. We examined the effects of mutations in and around these signals on the basolateral targeting of rat FcRn in rat inner medullary collecting duct cells. We also studied a second acidic cluster, Glu331/Glu333, some distance from either endocytosis signal. Some amino acid substitutions in the W–2 and W+3 positions disrupted the tryptophan-based basolateral-targeting signal without impairing its function in endocytosis. The tryptophan-based basolateral targeting and endocytosis signals are thus distinct but overlapping, as has been seen for collinear tyrosine-based signals. Surprisingly, the tryptophan-based basolateral-targeting signal required the aspartate pair of the dileucine-based signal. This acidic cluster, separated by two amino acids from the Φ residue of the tryptophan signal, is therefore a component of both of the basolateral-targeting signals. The acidic cluster Glu-331/Glu333 was not required for basolateral targeting, but its replacement reduced endocytosis.

Administration of therapeutic proteins by methods other than injection is limited, in part, by inefficient penetration of epithelial barriers. Therefore, unique approaches to breaching these barriers are needed. The neonatal constant region fragment (Fc) receptor (FcRn), which is responsible for IgG transport across the intestinal epithelium in newborn rodents, is expressed in epithelial cells in adult humans and non-human primates. Here we show that FcRn-mediated transport is functional in the lung of non-human primates and that this transport system can be used to deliver erythropoietin (Epo) when it is conjugated to the Fc domain of IgG1. FcRn-dependent absorption was more efficient when the EpoFc fusion protein was deposited predominantly in the upper and central airways of the lung, where epithelial expression of FcRn was most prominently detected. To optimize fusion protein absorption in the lung, we created a recombinant "monomeric-Epo" Fc fusion protein comprised of a single molecule of Epo conjugated to a dimeric Fc. This fusion protein exhibited enhanced pharmacokinetic and pharmacodynamic properties. The bioavailability of the EpoFc monomer when delivered through the lung was approximately equal to that reported for unconjugated Epo delivered s.c. in humans. These studies show that FcRn can be harnessed to noninvasively deliver bioactive proteins into the systemic circulation in therapeutic quantities.

Administration of therapeutic proteins by methods other than injection is limited, in part, by inefficient penetration of epithelial barriers. Therefore, unique approaches to breaching these barriers are needed. The neonatal constant region fragment (Fc) receptor (FcRn), which is responsible for IgG transport across the intestinal epithelium in newborn rodents, is expressed in epithelial cells in adult humans and non-human primates. Here we show that FcRn-mediated transport is functional in the lung of non-human primates and that this transport system can be used to deliver erythropoietin (Epo) when it is conjugated to the Fc domain of lgG1. FcRn-dependent absorption was more efficient when the EpoFc fusion protein was deposited predominantly in the upper and central airways of the lung, where epithelial expression of FcRn was most prominently detected. To optimize fusion protein absorption in the lung, we created a recombinant "monomeric-Epo" Fc fusion protein comprised of a single molecule of Epo conjugated to a dimeric Fc. This fusion protein exhibited enhanced pharmacokinetic and pharmacodynamic properties. The bioavailability of the EpoFc monomer when delivered through the lung was approximately equal to that reported for unconjugated Epo delivered s.c. in humans. These studies show that FcRn can be harnessed to noninvasively deliver bioactive proteins into the systemic circulation in therapeutic quantities.

Endocytosis of membrane proteins is typically mediated by signals present in their cytoplasmic domains. These signals usually contain an essential tyrosine or pair of leucine residues. Both tyrosine- and dileucine-based endocytosis signals are recognized by the adaptor complex AP-2. The best understood of these interactions occurs between the tyrosine-based motif, YXXΦ, and the μ2 subunit of AP-2. We recently reported a tryptophan-based endocytosis signal, WLSL, contained within the cytoplasmic domain of the neonatal Fc receptor. This signal resembles YXXΦ. We have investigated the mechanism by which the tryptophan-based signal is recognized. Both interaction assays in vitro and endocytosis assays in vivo show that μ2 binds the tryptophan-based signal. Furthermore, the WLSL sequence binds the same site as YXXΦ. Unlike the WXXF motif, contained in stonin 2 and other endocytic proteins, WLSL does not bind the α subunit of AP-2. These observations reveal a functional similarity between the tryptophan-based endocytosis signal and the YXXΦ motif, and an unexpected versatility of μ2 function.

Maternal antibodies transported across the placenta protect the newborn. Maternal immunoglobulin G (IgG) concentrations in fetal blood increase from early in the second trimester through term, most antibodies being acquired during the third trimester. IgG1 is the most efficiently transported subclass and IgG2 the least. Transfer across the syncytiotrophoblast of the chorionic villi is mediated by the neonatal Fc receptor, FcRn. Immune complexes are absorbed in the stroma of the villi, probably by FcgammaRI, FcgammaRII, and FcgammaRIII on placental macrophages. The mechanism of IgG transport across the endothelium of fetal capillaries is not understood. Endothelial cells in terminal villi express FcgammaRIIb. However, it is not known whether this receptor transports IgG or prevents transport of immune complexes to the fetus.

The intestinal Fc receptor, FcRn, functions in the maternofetal transfer of gamma globulin (IgG) in the neonatal rodent. In humans, most of this transfer is presumed to occur in utero via the placenta. Although the fetus swallows amniotic fluid that contains immunoglobulin, it is unknown whether this transfer also occurs via the fetal intestine. A human FcRn has been identified in the syncytiotrophoblast that mediates the maternofetal transfer of antibody. It has also been identified in human fetal intestine and is postulated to function in IgG transport. We hypothesize that the human fetal intestinal FcRn may play a role in IgG transport from the amniotic fluid into the fetal circulation. The aim of this study was to characterize the distribution of the FcRn along the human fetal intestine. Lysates prepared from human fetal intestine and from a nonmalignant human fetal intestinal epithelial cell line (H4) were subjected to Western blot analysis and probed using anti-FcRn antibodies. A 42-kD band, consistent with the known molecular weight of the FcRn, was detected along the human fetal intestine and in H4 cells. Expression of the human FcRn was confirmed with immunohistochemistry. Our study demonstrates the expression of FcRn along the human fetal intestine and in a human nonmalignant fetal intestinal epithelial cell line (H4), which by location indicates that FcRn could play a role in the uptake and transport of IgG in the human fetus.

The neonatal Fc receptor, FcRn, transports immunoglobulin G (IgG) across intestinal epithelial cells of suckling rats and mice from the lumenal surface to the serosal surface. In cell culture models FcRn transports IgG bidirectionally, but there are differences in the mechanisms of transport in the two directions. We investigated the effects of mutations in the cytoplasmic domain of FcRn on apical to basolateral and basolateral to apical transport of Fc across rat inner medullary collecting duct (IMCD) cells. Basolateral to apical transport did not depend upon determinants in the cytoplasmic domain. In contrast, an essentially tailless FcRn was markedly impaired in apical to basolateral transport. Using truncation and substitution mutants, we identified serine-313 and serine-319 as phosphorylation sites in the cytoplasmic domain of FcRn expressed in Rat1 fibroblasts. Mutations at Ser-319 did not affect transcytosis across IMCD cells. FcRn-S313A was impaired in apical to basolateral transcytosis to the same extent as tailless FcRn, whereas FcRn-S313D transported at wild-type levels. FcRn-S313A recycled more Fc to the apical medium than the wild-type receptor, suggesting that Ser-313 is required to allow FcRn to be diverted from an apical recycling pathway to a transcytotic pathway.

The neonatal Fc receptor, FcRn, transports immunoglobulin G across intestinal cells in suckling rats. FcRn enters these cells by endocytosis and is present on the apical and basolateral surfaces. We investigated the roles of aromatic amino acids and a dileucine motif in the cytoplasmic domain of rat FcRn. We expressed mutant FcRn in which alanine replaced Trp-311, Leu-322, and Leu-323, or Phe-340 in the inner medullary collecting duct cell line IMCD. Individual replacement of the aromatic amino acids or the dileucine motif only partially blocked endocytosis of (125)I-Fc, whereas uptake by FcRn containing alanine residues in place of both Trp-311 and the dileucine motif was reduced to the level obtained with the tailless receptor. Leu-314 was required for the function of the tryptophan-based endocytosis signal, and Asp-317 and Asp-318 were required for the dileucine-based signal. Nonvectorial delivery of newly synthesized FcRn to the two cell surfaces was unaffected by loss of the endocytosis signals. However, the steady-state distribution of endocytosis mutants was predominantly apical, unlike wild-type FcRn, which was predominantly basolateral. This shift appeared to arise because the loss of endocytosis signals inhibited apical to basolateral transcytosis of FcRn more than basolateral to apical transcytosis.

The neonatal Fc receptor, FcRn, is expressed in human placental syncytiotrophoblast, capillary endothelium, intestinal epithelium, and other tissues. By analogy with its role in the mouse, human FcRn is expected to transport maternal IgG to the foetus, and protect circulating IgG from catabolism. The larger subunit of FcRn is homologous to the a chains of the major histocompatibility complex (MHC) class I proteins, but is encoded outside the MHC on chromosome 19. We report the isolation of clones encoding the a chain of human FcRn from chromosome 19-specific libraries. The sequence revealed a similar organization to classical and non-classical MHC, and MHC-related genes. Compared with classical MHC class I genes, the human FcRn a chain gene has expanded by acquiring many repetitive sequences in its introns, including multiple Alu elements in the fourth intron. Primer extension analysis showed that there are two transcription initiation sites in the upstream flanking sequence.

The neonatal Fc receptor, FcRn, transports immunoglobulin G (IgG) across cellular barriers between mother and offspring. FcRn also protects circulating IgG from catabolism, probably during transport across the capillary endothelium. Only one cell culture model of transcytosis has been used extensively, the transport of IgA from the basolateral to the apical surface of Madin-Darby canine kidney cells by the polymeric immunoglobulin receptor (pIgR). We report that rat inner medullary collecting duct (IMCD) cells transfected with DNA encoding the (alpha) subunit of rat FcRn specifically and saturably transport Fc when grown as polarized monolayers. Using this system, we have found that transcytosis by FcRn, like transcytosis by the pIgR, depends upon an intact microtubule system. FcRn differs most strikingly from the pIgR in its ability to transport its ligand in both the apical to basolateral and basolateral to apical directions. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibited basolateral to apical transport by FcRn more than apical to basolateral transport, suggesting that there are differences in the mechanisms of transport in the two directions. Lastly, we found that transcytosis by FcRn depends upon vesicular acidification. We anticipate that the IMCD cell culture model will allow further elucidation of the mechanism of IgG transport by FcRn.

The 5′-flanking region of the human FcRn α-chain gene was analyzed for its ability to directly express the chloramphenicol acetyltransferase (CAT) reporter gene in NIH3T3 and Lu106 cells. Transient transfection of the CAT constructs revealed that there was promoter activity in the region −660 to +300 of the 5′-flanking sequence. Electrophoretic mobility-shift assays showed that there are functional binding sites for Sp1 or Sp1-like factors, AP1 or a related factor, and additional unidentified proteins in the promoter region.