Bonny L. Dickinson

Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line

The MHC class I-related Fc receptor, FcRn, mediates the intestinal absorption of maternal IgG in neonatal rodents and the transplacental transport of maternal IgG in humans by receptor-mediated transcytosis. In mice and rats, expression of FcRn in intestinal epithelial cells is limited to the suckling period. We have recently observed, however, clear expression of FcRn in the adult human intestine, suggesting a function for FcRn in intestinal IgG transport beyond neonatal life in humans. We tested this hypothesis using the polarized human intestinal T84 cell line as a model epithelium. Immunocytochemical data show that FcRn is present in T84 cells in a punctate apical pattern similar to that found in human small intestinal enterocytes. Solute flux studies show that FcRn transports IgG across T84 monolayers by receptor-mediated transcytosis. Transport is bidirectional, specific for FcRn, and dependent upon endosomal acidification. These data define a novel bidirectional mechanism of IgG transport across epithelial barriers that predicts an important effect of FcRn on IgG function in immune surveillance and host defense at mucosal surfaces.

Receptor-mediated immunoglobulin G transport across mucosal barriers in adult life : functional expression of FcRn in the mammalian lung

Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain the immunoglobulins (Ig)G and secretory IgA (sIgA) that function together in host defense. Exactly how IgG crosses epithelial barriers to function in mucosal immunity remains unknown. Here, we test the idea that the MHC class I–related Fc-receptor, FcRn, transports IgG across the mucosal surface of the human and mouse lung from lumen to serosa. We find that bronchial epithelial cells of the human, nonhuman primate, and mouse, express FcRn in adult-life, and demonstrate FcRn-dependent absorption of a bioactive Fc-fusion protein across the respiratory epithelium of the mouse in vivo. Thus, IgG, like dimeric IgA, can cross epithelial barriers by receptor-mediated transcytosis in adult animals. These data show that mucosal surfaces that express FcRn reabsorb IgG and explain a mechanism by which IgG may act in immune surveillance to retrieve lumenal antigens for processing in the lamina propria or systemically.

MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells.

The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the β2-microglobulin (β2m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical FcγRs: FcγRI, FcγRII, and FcγRIII.

The antifungal antibiotic, clotrimazole, inhibits chloride secretion by human intestinal T84 cells via blockade of distinct basolateral K+ conductances. Demonstration of efficacy in intact rabbit colon and in an in vivo mouse model of cholera.

The antifungal antibiotic clotrimazole (CLT) blocks directly and with high potency the Ca2+-activated K+ channels of human erythrocytes, erythroleukemia cells, and ferret vascular smooth muscle cells. We recently reported that CLT inhibits Cl- secretion in human intestinal T84 cells, likely by affecting K+ transport (Rufo, P.A., L. Jiang, S.J. Moe, C. Brugnara, S.L. Alper, and W.I. Lencer. 1996. J. Clin. Invest. 98:2066-2075). To determine if CLT had direct effects on K+ conductances in T84 cells, we selectively permeabilized apical membranes of confluent T84 cell monolayers using the ionophore amphotericin B. This technique permits direct measurement of basolateral K+ transport. We found that CLT and a stable des-imidazolyl derivative inhibited directly two pharmacologically distinct basolateral membrane K+conductances, but had no effect on apical membrane Cl- conductances. The effects of CLT on Cl- secretion were also examined in intact tissue. CLT inhibited forskolin-induced Cl- secretion in rabbit colonic mucosal sheets mounted in Ussing chambers by 91%. CLT also inhibited cholera toxin-induced intestinal Cl- secretion in intact mice by 94%. These data provide direct evidence that CLT blocks Cl- secretion in intestinal T84 cells by inhibition of basolateral K+ conductances, and show that CLT inhibits salt and water secretion from intact tissue in vitro and in vivo. The results further support the suggestion that CLT and its metabolites may show clinical efficacy in the treatment of secretory diarrheas of diverse etiologies.

Functional Reconstitution of Human FcRn in Madin-Darby Canine Kidney Cells Requires Co-expressed Human β2-Microglobulin

The major histocompatibility complex class I-related neonatal Fc receptor, FcRn, assembles as a heterodimer consisting of a heavy chain and β2-microglobulin (β2m), which is essential for FcRn function. We observed that, in Madin-Darby canine kidney (MDCK) cells, the function of human FcRn in mediating the bidirectional transport of IgG was significantly increased upon co-expression of the human isoform of β2m. In MDCK cells, the presence of human β2m endowed upon human FcRn an enhanced ability to exit the endoplasmic reticulum and acquire mature carbohydrate side-chain modifications at steady state, a faster kinetics of maturation, and augmented localization at the cell surface as a mature glycoprotein able to bind IgG. Although human FcRn with immature carbohydrate side-chain modifications was capable of exhibiting pH-dependent binding of IgG, only human FcRn with mature carbohydrate side-chain modifications was detected on the cell surface. These results show that human FcRn travels to the cell surface via the normal secretory pathway and that the appropriate expression and function of human FcRn in MDCK cells depends upon the co-expression of human β2m.

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.

Crosstalk Between PKA and Epac Regulates the Phenotypic Maturation and Function of Human Dendritic Cells

The cAMP-dependent signaling pathways that orchestrate dendritic cell (DC) maturation remain to be defined in detail. Although cAMP was previously thought to signal exclusively through protein kinase A (PKA), it is now clear that cAMP also activates exchange protein activated by cAMP (Epac), a second major cAMP effector. Whether cAMP signaling via PKA is sufficient to drive DC maturation or whether Epac plays a role has not been examined. In this study, we used cAMP analogs to selectively activate PKA or Epac in human monocyte-derived DCs and examined the effect of these signaling pathways on several hallmarks of DC maturation. We show that PKA activation induces DC maturation as evidenced by the increased cell-surface expression of MHC class II, costimulatory molecules, and the maturation marker CD83. PKA activation also reduces DC endocytosis and stimulates chemotaxis to the lymph node-associated chemokines CXCL12 and CCL21. Although PKA signaling largely suppresses cytokine production, the net effect of PKA activation translates to enhanced DC activation of allogeneic T cells. In contrast to the stimulatory effects of PKA, Epac signaling has no effect on DC maturation or function. Rather, Epac suppresses the effects of PKA when both pathways are activated simultaneously. These data reveal a previously unrecognized crosstalk between the PKA and Epac signaling pathways in DCs and raise the possibility that therapeutics targeting PKA may generate immunogenic DCs, whereas those that activate Epac may produce tolerogenic DCs capable of attenuating allergic or autoimmune disease.

The Cystine/Glutamate Antiporter Regulates Dendritic Cell Differentiation and Antigen Presentation

The major cellular antioxidant glutathione is depleted during HIV infection and in obesity. Although the consequence of glutathione depletion on immune function is starting to emerge, it is currently not known whether glutathione dysregulation influences the differentiation and maturation of dendritic cells (DCs). Moreover, the effect of glutathione depletion on DC effector functions, such as Ag presentation, is poorly understood. Glutathione synthesis depends on the cystine/glutamate antiporter, which transports the rate-limiting precursor cystine into the cell in exchange for glutamate. In this paper, we present a detailed study of antiporter function in DCs and demonstrate a role for the antiporter in DC differentiation and cross-presentation. We show that the antiporter is the major mechanism for transport of cystine and glutamate and modulates the intracellular glutathione content and glutathione efflux from DCs. Blocking antiporter-dependent cystine transport decreases intracellular glutathione levels, and these effects correlate with reduced transcription of the functional subunit of the antiporter. We further demonstrate that blocking antiporter activity interferes with DC differentiation from monocyte precursors, but antiporter activity is not required for LPS-induced phenotypic maturation. Finally, we show that inhibiting antiporter uptake of cystine interferes with presentation of exogenous Ag to class II MHC-restricted T cells and blocks cross-presentation on MHC class I. We conclude that aberrant antiporter function disrupts glutathione homeostasis in DCs and may contribute to impaired immunity in the diseased host.

The multiple roles of major histocompatibility complex class-I-like molecules in mucosal immune function

The human major histocompatibility complex (MHC) on chromosome 6 encodes three classical class-I genes: human leukocyte antigens (HLA) A, B, and C. These polymorphic genes encode a 43- to 45-kDa cell surface glycoprotein that, in association with the 12-kDa β2-microglobulin molecule, functions in the presentation of nine amino acid peptides to the T-cell receptor of CD8-bearing T lymphocytes and killer inhibitory receptors on natural killer cells. In addition to these ubiquitously expressed, polymorphic proteins, the human genome also encodes several nonclassical MHC class-I-like, or class Ib, genes that, in general, encode nonpolymorphic molecules involved in various specific immunological functions. Many of these genes, including CD1, the neonatal Fc receptor for IgG, HLA-G, HLA-E, the MHC class-I chainrelated gene A, and Hfe, are prominently displayed on epithelial cells, suggesting an important role in epithelial cell biology.

The cystine/glutamate antiporter regulates the functional expression of indoleamine 2,3-dioxygenase in human dendritic cells.

To the Editor: The cystine ⁄ glutamate antiporter controls the biosynthesis of the major cellular antioxidant glutathione (GSH) by transporting cystine, the rate-limiting precursor of GSH synthesis, into the cell in exchange for glutamate [1]. We recently demonstrated that blocking the function of the antiporter depleted GSH and significantly impaired dendritic cell (DC) presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway [2]. To explain this defect, here we test the hypothesis that the antiporter regulates DC expression of indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme that has emerged as a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are largely mediated by DCs, a detailed understanding of how IDO is regulated in these cells is critical for the design of targeted strategies to induce robust immune tolerance. To quantify changes in IDO mRNA transcript levels following antiporter blockade, we used digital mRNA profiling and compared immature DCs, DCs matured by incubation with LPS (mature DCs) and mature DCs cultured in cystine ⁄ cysteine-free medium to prevent antiporter uptake of cystine [see online Supporting Information for Materials and Methods]. Consistent with the observation of others, we found that IDO mRNA transcripts were increased in mature DCs relative to immature DCs [3] (Fig. 1A). In line with our hypothesis, IDO mRNA transcripts were significantly increased in mature DCs cultured in cystine ⁄ cysteine-free medium when compared to both immature and mature DCs (Fig. 1A). As IDO is tightly regulated at the translational and post-translational levels, we next quantified kynurenine levels in DC culture supernatants as a measure of IDO enzymatic activity. To do this, we used a well-established colorimetric method [4]. Antiporter uptake of cystine was inhibited by culturing DCs in cystine ⁄ cysteine-free medium for 16 and 24 h or by treating DCs with L-homocysteic acid (LHC), a potent competitive inhibitor of the antiporter that does not serve as a substrate for GSH synthesis [5]. While immature DCs did not exhibit IDO enzymatic activity (not shown), LPS induced a modest increase in IDO activity as previously reported [3] (Fig. 1B). In line with our mRNA data, IDO enzymatic activity was significantly increased when antiporterdependent uptake of cystine was inhibited (Fig. 1B). Both LHC and cystine ⁄ cysteine-free medium significantly increased IDO enzymatic activity at 16 and 24 h relative