Activation of the JAK/STAT-1 signaling pathway by IFN-gamma can down-regulate functional expression of the MHC class I-related neonatal Fc receptor for IgG.

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.

Identification and characterization of an alternatively spliced variant of the MHC class I-related porcine neonatal Fc receptor for IgG.

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 0.795kb) 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 1.071kb cDNA encodes the full-length pFcRn (pFcRn-L), whereas the 0.795kb cDNA codes for a truncated pFcRn (pFcRn-S) with deletion of 92 amino acids matching the alpha2 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 beta(2)-microglobulin (beta(2)m). 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.

Substituted organosiloxanes as potential therapeutics for treatment and prevention of neurodegenerative diseases.

Extensive testing of hydrolysates of commercially available organosilanes has identified a number of bifunctional organosiloxane compounds that show potential as therapeutics for treatment of diseases characterized by amyloid deposition such as Alzheimer's disease (AD). All of these compounds protect from and/or reverse the metal-induced aggregation of amyloid Abeta(1-42) peptide in dynamic light scattering (DLS) assays in trifluoroethanol (TFE) solutions, protect from and/or reverse the metal-induced loss of alpha-helical structure in TFE solutions of amyloid Abeta(1-42) as measured by circular dichroism (CD), and are able to cross blood-brain barrier models at rates above background using Caco-2 and MDCK cell permeation assays. Based on these studies, we conclude that members of this class of bifunctional organosiloxanes are promising candidates for testing in treatment and/or prevention of AD and other diseases characterized by amyloid deposition.

Characterization of basolateral-targeting signals in the neonatal Fc receptor.

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 YXXPhi 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 Phi 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.

Recognition of the tryptophan-based endocytosis signal in the neonatal Fc Receptor by the mu subunit of adaptor protein-2.

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, YXXPhi, and the mu2 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 YXXPhi. 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 mu2 binds the tryptophan-based signal. Furthermore, the WLSL sequence binds the same site as YXXPhi. Unlike the WXXF motif, contained in stonin 2 and other endocytic proteins, WLSL does not bind the alpha subunit of AP-2. These observations reveal a functional similarity between the tryptophan-based endocytosis signal and the YXXPhi motif, and an unexpected versatility of mu2 function.

Pulmonary delivery of an erythropoietin Fc fusion protein in non-human primates through an immunoglobulin transport pathway.

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.

Placental transport of immunoglobulin G.

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.

Distribution of the IgG Fc receptor, FcRn, in the human fetal intestine.

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.